Beta-carotene Biosynthesis Research Articles

Fixed vs. variable light quality in vertical farming: Impacts on vegetative growth and nutritional quality of lettuce.

Lettuce (Lactuca sativa) is commonly produced in vertical farms. The levels of nutritionally important phytochemicals such as beta-carotene (precursor to vitamin A) are generally low in lettuce. In this study, we investigated the benefits of variable lighting strategy (i.e., varying the light quality during production) on maintaining plant growth and increasing the biosynthesis of beta-carotene and anthocyanin. We tested two variable lighting methods, using green and red romaine lettuce, namely (i) providing growth lighting (supports vegetative growth) initially (21 days) followed by a high percentage of blue light (supports biosynthesis of phytochemicals) at final stages (10 days) and (ii) providing a high percentage of blue light initially followed by growth lighting at final stages. Our results indicate that the variable lighting method with initial growth lighting and high percentage of blue at final stages can maintain vegetative growth and enhance phytochemicals such as beta-carotene in green romaine lettuce while both variable lighting methods were not effective in red romaine lettuce. In green romaine lettuce, we did not observe a significant reduction in shoot dry weight but there was an increase in beta-carotene (35.7%) in the variable compared to the fixed lighting method with growth lighting for the entire duration. The physiological bases for differences in vegetative growth and synthesis of beta-carotene and anthocyanin in the variable and fixed lighting methods are discussed.

Methylotroph bacteria and cellular metabolite carotenoid alleviate ultraviolet radiation-driven abiotic stress in plants.

Increasing UV radiation in the atmosphere due to the depletion of ozone layer is emerging abiotic stress for agriculture. Although plants have evolved to adapt to UV radiation through different mechanisms, but the role of phyllosphere microorganisms in counteracting UV radiation is not well studied. The current experiment was undertaken to evaluate the role of phyllosphere Methylobacteria and its metabolite in the alleviation of abiotic stress rendered by ultraviolet (UV) radiation. A potential pink pigmenting methylotroph bacterium was isolated from the phylloplane of the rice plant (oryzae sativa). The 16S rRNA gene sequence of the bacterium was homologous to the Methylobacter sp. The isolate referred to as Methylobacter sp N39, produced beta-carotene at a rate (μg ml-1 d-1) of 0.45-3.09. Biosynthesis of beta-carotene was stimulated by brief exposure to UV for 10 min per 2 days. Carotenoid biosynthesis was predicted as y = 3.09 × incubation period + 22.151 (r 2 = 0.90). The carotenoid extract of N39 protected E. coli from UV radiation by declining its death rate from 14.67% min-1 to 4.30% min-1 under UV radiation. Application of N39 cells and carotenoid extract also protected rhizobium (Bradyrhizobium japonicum) cells from UV radiation. Scanning electron microscopy indicated that the carotenoid extracts protected E. coli cells from UV radiation. Foliar application of either N39 cells or carotenoid extract enhanced the plant's (Pigeon pea) resistance to UV irradiation. This study highlight that Methylobacter sp N39 and its carotenoid extract can be explored to manage UV radiation stress in agriculture.

Dynamics of the gut microbiota in rats after hypobaric hypoxia exposure.

BackgroundGut microbiota plays an important role in host health and is influenced by multiple factors. Hypobaric hypoxia usually existing at high altitude conditions can adversely affect normal physiological functions. However, the dynamic changes of gut microbiota influenced by hypobaric hypoxia have not been elucidated.MethodsIn this study, we collected fecal samples from seven rats at 14 time points from entering the hypobaric chamber (eight time points) to leaving the chamber (six time points) and five rats served as normoxic controls. Metagenome sequencing was performed on all samples and the dynamics of taxa and functions were analyzed.ResultsWe found that the α-diversity was changed in the first 5 days after entering or leaving the hypobaric chamber. The β-diversity analysis revealed that gut microbiota structure was significantly separated among 14 time points. After entering the chamber, the relative abundance of Bacteroides decreased and the most abundant genus turned into Prevotella. The abundance of Firmicutes and Bacteroidetes showed an opposite trend and both have a significant change within 5 days after entering or leaving the hypobaric hypoxia chamber. Some obligate anaerobic bacteria belonging to Desulfovibrio and Alistipes were significantly enriched after entering the chamber for 5 weeks, whereas Probiotics like Bifidobacterium and Lactococcus, and short-chain fatty acids producers like Butyrivibrio and Pseudobutyrivibrio were significantly enriched after leaving the chamber for 3 weeks. Microbial functions like ‘Two-component regulatory system’, ‘beta-carotene biosynthesis’ and ‘Fatty acid biosynthesis’ were significantly enriched after entering the chamber for 5 weeks. Hypobaric hypoxia conditions could deeply affect the diversity and structure of gut microbiota. The alterations of abundance of dominant taxa (Firmicutes and Bacteroidetes), increased anaerobes and decreased probiotics induced by hypobaric hypoxia conditions might affect the host health.

Comparative transcriptome profiling of different tissues from beta-carotene-enhanced transgenic soybean and its non-transgenic counterpart

Beta-carotene-enhanced transgenic soybeans, harboring genes encoding phytoene synthase and carotene desaturase under the control of a seed-specific promoter, were developed to alleviate vitamin A deficiency in populations, the diet of which was deficient in this vitamin. However, metabolic engineering of carotenoid biosynthetic pathways often has unintended effects, leading to major metabolic changes in plants that harbor endogenous beta-carotene biosynthesis pathways. In the present study, we performed transcriptome profiling analysis using RNA-seq to investigate the changes in the transcriptome and some unintended pleiotropic effects on the leaves, stems, roots, and seeds of beta-carotene-enhanced transgenic soybean lines, and compared them to those of their non-transgenic counterpart donor variety Kwangan. We observed that transgenic soybeans showed significant changes in secondary metabolic biosynthesis in leaves and down-regulated galactose metabolism in roots. Differentially expressed genes in the transgenic group, which were significantly up-regulated, included those encoding glycine-aspartic acid-serine-leucine-motif esterase/lipase, known as cutin synthase and cutinase. These results suggested enhanced beta-carotene biosynthesis may affect related enzymes to carbohydrate metabolism and fatty acid metabolism. Hence, we speculated that upregulation of cutin polymerization resulted in thickened seed coat and delayed seed germination of transgenic soybeans. Furthermore, downregulation of raffinose family oligosaccharide biosynthesis may cause redundancy of myo-inositol, a substrate of phytin formation. This could lead to phytic globoids accumulation in transgenic soybean seeds. The present imformation would be important for transgenic plant development via carotenoid metabolic engineering, with focus on beta-carotene over-production.

Light regulates a Phycomyces blakesleeanus gene family similar to the carotenogenic repressor gene of Mucor circinelloides

The transcription of about 5–10 % of the genes in Phycomyces blakesleeanus is regulated by light. Among the most up-regulated, we have identified four genes, crgA–D, with similarity to crgA of Mucor circinelloides, a gene encoding a repressor of light-inducible carotenogenesis. The four proteins have the same structure with two RING RING Finger domains and a LON domain, suggesting that they could act as ubiquitin ligases, as their M. circinelloides homolog. The expression of these genes is induced by light with different thresholds as in other Mucoromycotina fungi like Blakeslea trispora and M. circinelloides. Only the P. blakesleeanus crgD gene could restore the wild type phenotype in a M. circinelloides null crgA mutant suggesting that P. blakesleeanus crgD is the functional homolog of crgA in M. circinelloides. Despite their sequence similarity it is possible that the P. blakesleeanus Crg proteins do not participate in the regulation of beta-carotene biosynthesis since none of the carotene-overproducing mutants of P. blakesleeanus had mutations in any of the crg genes. Our results provide further support of the differences in the regulation of the biosynthesis of beta-carotene in these two Mucoromycotina fungi.

Lipid production in Dunaliella bardawil under autotrophic, heterotrophic and mixotrophic conditions

ABSTRACT Many microalgae are rich in lipid. Due to their low growth rates in the autotrophic culture, the best alternative is to cultivate cells under different conditions such as heterotrophic or mixotrophic, which results in the highest yield of biomass and lipid in the shortest duration. In this study, Dunaliela bardawil (UTEX 2538) green microalgae was cultivated under different culture conditions, autotrophic, heterotrophic and mixotrophic, and effects on cell concentration, lipid production and reactive oxygen species (ROS), total chlorophyll and beta-carotene concentrations were investigated. Due to very low cellular growth, this alga is not recommended for heterotrophic culture. In terms of mixotrophic conditions containing different concentrations of glucose or acetate, the highest cell growth and lipid production in 60mM glucose was similar to the control (autotrophic condition), while the concentrations of chlorophyll and beta-carotene decreased. However, at all concentrations of acetate, a slight increase in cell growth was achieved, while the lipid content decreased. Additionally, the concentrations of chlorophyll and beta-carotene increased. A positive correlation was observed between beta-carotene biosynthesis and lipid production, as well as levels of reactive species of oxygen and lipid production in the presence of glucose and acetate. This study showed that for D. bardawil the mixotrophic culture with 60mM glucose was the most suitable type of culture for increasing lipid content and cell growth rates in less time (one week).

Expression Differences of Pigment Structural Genes and Transcription Factors Explain Flesh Coloration in Three Contrasting Kiwifruit Cultivars.

Fruits of kiwifruit cultivars (Actinidia chinensis and A. deliciosa) generally have green or yellow flesh when ripe. A small number of genotypes have red flesh but this coloration is usually restricted to the inner pericarp. Three kiwifruit cultivars having red (‘Hongyang’), or yellow (‘Jinnong-2’), or green (‘Hayward’) flesh were investigated for their color characteristics and pigment contents during development and ripening. The results show the yellow of the ‘Jinnong-2’ fruit is due to the combined effects of chlorophyll degradation and of beta-carotene accumulation. The red inner pericarps of ‘Hongyang’ fruit are due to anthocyanin accumulation. Expression differences of the pathway genes in the inner pericarps of the three different kiwifruits suggest that stay-green (SGR) controls the degradation of chlorophylls, while lycopene beta-cyclase (LCY-β) controls the biosynthesis of beta-carotene. The abundance of anthocyanin in the inner pericarps of the ‘Hongyang’ fruit is the results of high expressions of UDP flavonoid glycosyltransferases (UFGT). At the same time, expressions of anthocyanin transcription factors show that AcMYBF110 expression parallels changes in anthocyanin concentration, so seems to be a key R2R3 MYB, regulating anthocyanin biosynthesis. Further, transient color assays reveal that AcMYBF110 can autonomously induce anthocyanin accumulation in Nicotiana tabacum leaves by activating the transcription of dihydroflavonol 4-reductase (NtDFR), anthocyanidin synthase (NtANS) and NtUFGT. For basic helix-loop-helix proteins (bHLHs) and WD-repeat proteins (WD40s), expression differences show these may depend on AcMYBF110 forming a MYB-bHLH-WD40 complex to regulate anthocyanin biosynthesis, instead of it having a direct involvement.

Enhancing beta-carotene biosynthesis and gene transcriptional regulation in Blakeslea trispora with sodium acetate

The mold Blakeslea trispora is one of the most promising and economically attractive natural sources of β-carotene. In this study, the effects of sodium acetate (NaAC) on β-carotene content and production in mated B. trispora were investigated. The results show that the addition of acetate into mated medium could enhance β-carotene accumulation in B. trispora. The highest β-carotene content (59.91mg/g dry biomass) and production (2130mg/l) were obtained as adding 35mM of NaAC at stationary phase, which were 77.7% and 80.5% increments compared with that of the control, respectively. Furthermore, the effects of NaAC on expression levels of carotenogenesis genes in mated B. trispora were investigated. The results show that addition of NaAC in mated medium caused the induction of five carotenogenesis genes expression (hmgR, carG, ipi, carRA, and carB) and promoted de novo synthesis of β-carotene. The induction of five genes expression exhibited sequential gene expression profiles and the five gene expression were ranging from 1.8 to 3.8 folds increment as early as 24h after NaAC addition. We demonstrate that NaAC stimulation of β-carotene biosynthesis in mated B. trispora involved in change at genes transcriptional levels. Such regulatory mechanism provides an explanation for effect of NaAC on the biosynthesis of β-carotene in mated B. trispora.

Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Do cytokinins, volatile isoprenoids and carotenoids synergically delay leaf senescence?

Photo-Isomerization of Prolycopene in Chloroplast Occurs through the Triplet State: Implications on Carotenoid Biosynthesis

The carotenoid lycopene in photosynthetic organisms forms the critical precursor to the biosynthesis of reaction center carotenoids beta-carotene and lutein. In plants and cyanobacteria, a redox-controlled flavoenzyme carotenoid isomerase (CRTISO) catalyzes the four-step geometric isomerization of 7,9,9′,7′-tetra-cis-lycopene (prolycopene) to all-trans-lycopene. In chloroplasts, the functional loss of CRTISO has been shown to be rescued by a light-mediated isomerization pathway. In order to address the chloroplast-specificity and compare the efficiency of the photoisomerization reaction against redox-controlled enzyme catalysis, we need to track the excited state dynamics of prolycopene, and evaluate the nature of electronic states that lead to the photoisomerization. Using broadband femtosecond transient absorption spectroscopy, we observe ∼610 fs rise of the triplet state from the photoexcited S2 with a quantum yield of ∼0.19. The triplet state mediates the first C=C bond isomerization at symmetric 9 or 9′ position on the tetra-cis backbone to yield the tri-cis-lycopene with 15% quantum yield. However, direct sensitization of the photoreactive triplet state via meso-tetraphenyl porphyrin sensitizer under steady state illumination leads to all-trans-lycopene with 58% quantum efficiency. Our work implies that long-lived chlorophyll triplets activate the efficient isomerization of prolycopene in chloroplasts in absence of CRTISO activity. In presence of CRTISO, the role of redox equivalents in dark isomerization of prolycopene, and the light induced signaling mechanisms for the regulation of carotenoid biosynthesis will be discussed.

Golden Rice: An effective tool to overcome vitamin A deficiency

Golden rice is a form of rice with biosyn-thesis of beta-carotene (a form of vitamin A). It is a variety of rice (Oryza sativa) produced through genetic engineering to biosynthesize beta-carotene, a precursor of vitamin A, in the edible parts of rice. Beta-carotene gives rice its golden or yellow colouring. White rice, on the other hand, does not contain carotenoids (i.e. beta carotene) and therefore lacks that golden colour-ing. Golden rice differs from its parental strain by the addition of three beta-carotene biosynthesis genes. At the time of publication, rice was considered a significant breakthrough in biotech-nology, as the researchers had engineered an entire biosynthetic pathway. In 2005, a new variety called Golden Rice 2, which produces up to 23 times more beta-carotene than the original rice, was announced. Although rice was developed as a humanitarian tool, it has met with significant opposition from environmental and anti-globalization activists. Golden Rice was one of sev-en winners of the 2015 Patents for Humanity Awards by the United States Patent and Trademark Office.

Enhancing beta-carotene production inSaccharomyces cerevisiaeby metabolic engineering

Beta-carotene is known to exhibit a number of pharmacological and nutraceutical benefits to human health. Metabolic engineering of beta-carotene biosynthesis in Saccharomyces cerevisiae has been attracting the interest of many researchers. A previous work has shown that S.cerevisiae successfully integrated with phytoene synthase (crtYB) and phytoene desaturase (crtI) from Xanthophyllomyces dendrorhous could produce beta-carotene. In the present study, we achieved around 200% improvement in beta-carotene production in S.cerevisiae through specific site optimization of crtI and crtYB, in which five codons of crtI and eight codons of crtYB were rationally mutated. Furthermore, the effects of the truncated HMG-CoA reductase (tHMG1) from S.cerevisiae and HMG-CoA reductase (mva) from Staphylococcus aureus on the production of beta-carotene in S.cerevisiae were also evaluated. Our results indicated that mva from a prokaryotic organism might be more effective than tHMG1 for beta-carotene production in S.cerevisiae.

Synthesis of Coenzyme Q10 and β-carotene by Yeasts Isolated from Antarctic Soil and Lichen in Response to Ultraviolet and Visible Radiations

The effect of different doses of visible (Vis), ultraviolet-capital A, Cyrillic (UVA), and mixed light (UVA + Vis) upon coenzyme Q(10) (CoQ(10)) and beta-carotene synthesis and biomass yield by the Sporobolomyces salmonicolor AL(1), Cryptococcus albidus AS(55), Cryptococcus laurentii AS(56), and C. laurentii AS(58) strains isolated from Antarctic samples was investigated. The beta-carotene concentration in the red strain biomass increased by 52% under irradiation with 11 J/cm(2) Vis, and the CoQ(10) concentration rose by 37% in relation to the control quantity obtained through dark cultivation. Under irradiation with 6 J/cm(2) UVA, the S. salmonicolor AL(1) strain synthesized 15% more beta-carotene; C. albidus AS(55), 22%; C. laurentii AS(56), 44%; and C. laurentii AS(58), 35% in relation to the control quantity. Irradiation with a low UVcapital A, Cyrillic + Vis dose significantly stimulated beta-carotene biosynthesis by the strains of the Cryptococcus genus (87%, 138%, and 100%), whereas S. salmonicolor AL(1) increased the beta-carotene content to a smaller degree (55%). Higher doses of all three irradiation types inhibited beta-carotene accumulation. Vis suppressed CoQ(10) biosynthesis in the Cryptococcus strains, whereas UVcapital A, Cyrillic and UVcapital A, Cyrillic + Vis inhibited it in all four strains. The S. salmonicolor AL(1) strain pre-treated with 0.02 J/cm(2) UVA synthesized twice as much CoQ(10) and beta-carotene when cultivated in the presence of Vis light in an 11-J/cm(2) dose.

Functional analysis of multiple carotenogenic genes from Lycium barbarum and Gentiana lutea L. for their effects on β-carotene production in transgenic tobacco

Carotenoids are red, yellow and orange pigments, which are widely distributed in nature and are especially abundant in yellow-orange fruits and vegetables and dark green leafy vegetables. Carotenoids are essential for photosynthesis and photoprotection in plant life and also have different beneficial effects in humans and animals (van den Berg et al. 2000). For example, beta-carotene plays an essential role as the main dietary source of vitamin A. To obtain further insight into beta-carotene biosynthesis in two important economic plant species, Lycium barbarum and Gentiana lutea L., and to investigate and prioritize potential genetic engineering targets in the pathway, the effects of five carotenogenic genes from these two species, encoding proteins including geranylgeranyl diphosphate synthase, phytoene synthase and delta-carotene desaturase gene, lycopene beta-cyclase, lycopene epsilon-cyclase were functionally analyzed in transgenic tobacco (Nicotiana tabacum) plants. All transgenic tobacco plants constitutively expressing these genes showed enhanced beta-carotene contents in their leaves and flowers to different extents. The addictive effects of co-ordinate expression of double transgenes have also been investigated.

Screening and characterization of proteorhodopsin color-tuning mutations in Escherichia coli with endogenous retinal synthesis

Proteorhodopsin is photoactive 7-transmembrane protein, which uses all -trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.

Effects of high hydrostatic pressure on the growth and β‐carotene production of Rhodotorula glutinis

The effects of high hydrostatic pressure (HHP) on the biomass and beta-carotene biosynthesis of Rhodotorula glutinis R68 were studied. After treatment with five repeated cycles at 300 MPa for 15 min, the barotolerant mutant PR68 was obtained. After 72 h of culture, the biomass of mutant PR68 was 21.6 g/l, decreased by 8.5% compared to the parental strain R68, but its beta-carotene production reached 19.4 mg/l, increased by 52.8% compared to the parental strain R68. The result of restriction fragment length polymorphism (RFLP) analysis suggested that mutant strain PR68 was likely to change in nucleic acid level, and thus enhanced beta-carotene production in this strain as a result of gene mutation induced by HHP treatment.

Engineering the halophilic bacterium Halomonas elongata to produce β-carotene

Engineering halophilic bacteria to produce carotenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with beta-carotene the most prominent. With this target, we expressed the beta-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure beta-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY, crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of beta-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 microg per g of dry weight) in 2% NaCl. On the contrary, no beta-carotene was detected in 15% NaCl. Successful construction of the beta-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production.

Distinct white collar‐1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression.

Chromoplast Morphology and β-Carotene Accumulation during Postharvest Ripening of Mango Cv. ‘Tommy Atkins'

Accumulation of beta-carotene and trans-cis isomerization of ripening mango mesocarp were investigated as to concomitant ultrastructural changes. Proceeding postharvest ripening was shown by relevant starch degradation, tissue softening, and a rising sugar/acid ratio, resulting in a linear decrease (R (2) = 0.89) of a ripening index (RPI(KS)) with increasing ripening time. A modest accumulation of all-trans-beta-carotene and its cis isomers resulted in a slight pigmentation of the mango chromoplasts, because ambient temperatures of 18.2-19.5 degrees C provided suboptimal ripening conditions, affecting color development and beta-carotene biosynthesis. The ultrastructures of chromoplasts from mango mesocarp and carrot roots were comparatively studied by means of light and transmission electron microscopy. Irrespective of the ripening stage, mango chromoplasts showed numerous plastoglobuli varying in size and electron density. They comprised the main part of carotenoids, thus supporting the partial solubilization of the pigments in lipid droplets. However, because different pigment-carrying tubular membrane structures were also observed, mango chromoplasts were assigned to the globular and reticulotubular types, whereas the crystalline type was confirmed for carrot chromoplasts. The large portions of naturally occurring cis-beta-carotene in mango fruits contrasted with the predominance of the all-trans isomer characteristic of carrots, indicating that the nature of the structure where carotenoids are deposited and the physical state of the pigments are crucial for the stability of the all-trans configuration.

Marker‐free transgenic (MFT) near‐isogenic introgression lines (NIILs) of ‘golden’ indica rice (cv. IR64) with accumulation of provitamin A in the endosperm tissue

We have developed near-isogenic introgression lines (NIILs) of an elite indica rice cultivar (IR64) with the genes for beta-carotene biosynthesis from dihaploid (DH) derivatives of golden japonica rice (cv. T309). A careful analysis of the DH lines indicated the integration of the genes of interest [phytoene synthase (psy) and phytoene desaturase (crtI)] and the selectable marker gene (hygromycin phosphotransferase, hph) in two unlinked loci. During subsequent crossing, progenies could be obtained carrying only the locus with psy and crtI, which was segregated independently from the locus containing the hph gene during meiotic segregation. The NIILs (BC(2)F(2)) showed maximum similarity with the recurrent parent cultivar IR64. Further, progenies of two NIILs were devoid of any fragments beyond the left or right border, including the chloramphenicol acetyltransferase (cat) antibiotic resistance gene of the transformation vector. Spectrophotometric readings showed the accumulation of up to 1.06 microg total carotenoids, including beta-carotene, in 1 g of the endosperm. The accumulation of beta-carotene was also evident from the clearly visible yellow colour of the polished seeds.