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Rice is one of the plants proven to possess antioxidant, anti-inflammatory, anti-proliferative, and whitening properties, making it one of the most beneficial plants in this regard. This study aimed to introduce a novel natural cosmetic and pharmaceutical product based on rice callus as a source of active ingredients that can inhibit skin melanoma cell (B16F10) proliferation and brighten the skin. The 2,4-D hormone at concentrations of 1 µg/mL and 1.5 µg/mL was used to induce rice callus formation. Rice callus extracts were then prepared using aqueous and ethanolic solvents, with a concentration of 1 mg/mL used for characterization tests. To determine the optimal hormone concentration, the phenols/flavonoids, antioxidant activity, proteins, and carbohydrates in the extracts were measured. The optimal concentration of the hormone was found to be 1 µg/mL. Finally, the anti-melanocyte and skin-whitening activity of the extracts was assessed through measurements of their cytotoxicity and inhibition of melanin synthesis-related enzymes. Cellular cytotoxicity measurements revealed that the ethanolic extract induced more cytotoxicity than the aqueous extract, with IC50 values of 566.3 µg/mL for the ethanolic extract and 1327 µg/mL for the aqueous extract. Skin-whitening-related tests demonstrated that the extracts were 1.7 times more effective than arbutin in inhibiting factors that cause hyperpigmentation. The aqueous extract achieved 85% inhibition of melanin biosynthesis at a concentration of 3200 µg/mL, compared to 68% for the ethanolic extract and 50% for arbutin. Based on these findings, rice callus extract can be introduced as a new, effective substance for skin-lightening and anti-melanocyte products in cosmeceutical and pharmaceutical formulations. [ABSTRACT FROM AUTHOR]

Rice is one of the plants proven to possess antioxidant, anti-inflammatory, anti-proliferative, and whitening properties, making it one of the most beneficial plants in this regard. This study aimed to introduce a novel natural cosmetic and pharmaceutical product based on rice callus as a source of active ingredients that can inhibit skin melanoma cell (B16F10) proliferation and brighten the skin. The 2,4-D hormone at concentrations of 1 µg/mL and 1.5 µg/mL was used to induce rice callus formation. Rice callus extracts were then prepared using aqueous and ethanolic solvents, with a concentration of 1 mg/mL used for characterization tests. To determine the optimal hormone concentration, the phenols/flavonoids, antioxidant activity, proteins, and carbohydrates in the extracts were measured. The optimal concentration of the hormone was found to be 1 µg/mL. Finally, the anti-melanocyte and skin-whitening activity of the extracts was assessed through measurements of their cytotoxicity and inhibition of melanin synthesis-related enzymes. Cellular cytotoxicity measurements revealed that the ethanolic extract induced more cytotoxicity than the aqueous extract, with IC50 values of 566.3 µg/mL for the ethanolic extract and 1327 µg/mL for the aqueous extract. Skin-whitening-related tests demonstrated that the extracts were 1.7 times more effective than arbutin in inhibiting factors that cause hyperpigmentation. The aqueous extract achieved 85% inhibition of melanin biosynthesis at a concentration of 3200 µg/mL, compared to 68% for the ethanolic extract and 50% for arbutin. Based on these findings, rice callus extract can be introduced as a new, effective substance for skin-lightening and anti-melanocyte products in cosmeceutical and pharmaceutical formulations.

This study aimed to investigate the correlation between callus regeneration rate and the expression of several genes responsible for cytokinin response and ethylene biosynthesis in the Ciherang, Mentik Wangi Susu, Hwayoung and Tarabas rice varieties. The callus regeneration rate of each rice variety was in vitro tested using N6 media, while the gene expression during the callus regeneration stages was examined using quantitative real‐time PCR (qRT‐PCR). Our results showed that the callus of Ciherang and Mentik Wangi Susu showed earlier green spot formation that then turned brown at a later stage, resulting in a low regeneration rate. While Hwayoung and Tarabas showed late green spot formation, high shoot regeneration was observed in both calluses. Gene expression analysis of regeneration media‐grown calluses showed that two cytokinin‐responsive genes, OsRR2 and OsRR6, were highly expressed in the Ciherang and Hwayoung callus, respectively. We also observed that ethylene biosynthesis genes such as OsACS1 and OsACO1 were highly expressed in the Mentik Wangi Susu and Hwayoung callus, respectively. Moreover, the expression of OsBBM1 was high in Hwayoung and Tarabas. Thus, the positive correlation between the expression of cytokinin‐responsive and ethylene biosynthesis genes with somatic embryogenesis activity likely depends on the induction level of OsBBM1.

Simple Summary: Climate change is currently threatening agriculture all around the world, resulting in a lack of water and restricting the growth of plants, especially rice. Rice production decreases with the increase in temperature. An improvement in fundamental knowledge is necessary to comprehend plant adaptation mechanisms as responses to heat stress. Physiological and anatomical responses of Khao Dawk Mali 105 (KDML105) rice to artificial heat stress were studied. Our findings offer useful data for projects aimed at improving heat stress tolerance in rice to enhance long-term global food security. Global warming is a serious problem, with significant negative impacts on agricultural productivity. To better understand plant anatomical adaptation mechanisms as responses to heat stress, improved basic knowledge is required. This research studied the physiological and anatomical responses of Khao Dawk Mali 105 (KDML105) to artificial heat stress. Dehusked seeds were sterilized and cultured on Murashige and Skoog (MS) medium, supplemented with 3 mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D) for callus induction. The cultures were maintained at 25 °C and 35 °C for 4 weeks, while the other culture was treated with heat shock at 42 °C for 1 week before further incubation at 25 °C for 3 weeks. Results revealed that elevated temperatures (35 °C and 42 °C) adversely impacted seedling growth. Plant height, root length, leaf number per plant, fresh and dry weight, chlorophyll a, chlorophyll b and total chlorophyll content decreased after heat stress treatment, while malondialdehyde (MDA) and electrolyte leakage percentage significantly increased, compared to the control. Heat stress induced ROS accumulation, leading to lipid peroxidation and membrane instability. Principal component analysis (PCA) and hierarchical cluster analysis (HCA) results also confirmed negative correlations between MDA, electrolyte leakage and other parameters. MDA content and electrolyte leakage are effective indicators of heat stress in rice. Surface anatomical responses of rice seedlings to heat stress were studied but significant alterations were not observed, and heat stress had no significant negative effects on KDML105 calli. Size and mass of calli increased because heat stress stimulated gene expression that induced thermotolerance. Our results provide useful information for rice breeding and heat stress tolerance programs to benefit long-term global food security. [ABSTRACT FROM AUTHOR]

Abstract Background β-carotene (provitamin A) is an important target for biofortification of crops as a potential solution to the problem of vitamin A deficiency that is prevalent in developing countries. A previous report showed that dominant expression of splicing variants in the Orange (Or) gene causes β-carotene accumulation in cauliflower curd. In this study, we focused on a putative orthologue of the cauliflower or gene in rice, Osor, and attempt to accumulate β-carotene in rice callus by modification of the Osor gene via genome editing using CRISPR/Cas9. Findings CRISPR/Cas9 vectors for the Osor gene were constructed and transformed into rice calli. Some transformed calli showed orange color due to β-carotene hyper-accumulation. Molecular analyses suggest that orange-colored calli are due to an abundance of in-frame aberrant Osor transcripts, whereas out-of-frame mutations were not associated with orange color. Conclusions We demonstrate that directed gene modification of the Osor gene via CRISPR/Cas9-mediated genome editing results in β-carotene fortification in rice calli. To date, golden rice, which accumulates β-carotene in rice endosperm, has been developed by conventional transgenic approaches. Our results suggest an alternative approach to enhancing β-carotene accumulation in crops.

Purpose: Plant residues are one of the main sources of soil organic matter in paddy fields, and elucidation of the bacterial communities decomposing plant residues was important to understand their function and roles, as the microbial decomposition of plant residues is linked to soil fertility. We conducted a DNA stable isotope probing (SIP) experiment to elucidate the bacterial community assimilating 13-carbon (C) derived from plant residue under an anoxic soil condition. In addition, we compared the bacterial community with that under the oxic soil condition, which was elucidated in our previous study (Lee et al. in Soil Biol Biochem 43:814-822, 2011). Materials and methods: We used the C-labeled dried rice callus cells as a model of rice plant residue. A paddy field soil was incubated with unlabeled and C-labeled callus cells. DNA extracted from the soils was subjected to buoyant density gradient centrifugation to fractionate C-enriched DNA. Then, polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) analysis of bacterial 16S rDNA band patterns and band sequencing method were used to evaluate bacterial community. Results and discussion: DGGE analysis showed that the band patterns in the C-enriched fractions were distinctly changed over time, while the changes in the community structure before fractionation were minor. Sequencing of the C-labeled DGGE bands revealed that Clostridia were a major group in the bacterial communities incorporating the callus-derived carbon although Gram-negative bacteria, and Actinobacteria also participated in the carbon flow from the callus under the anoxic condition. The proportion of Gram-negative bacteria and Actinobacteria increased on 14 days after the onset of incubation, suggesting that the callus was decomposed by diverse bacterial members on this phase. When the bacterial groups incorporating the C were compared between under anoxic and oxic soil conditions, the composition was largely different under the two opposite conditions. However, some members of Gram-negative bacteria were commonly found under the anoxic and oxic soil conditions. Conclusions: The majority of bacterial members assimilating the callus carbon was Clostridia in the soil under anoxic conditions. However, several Gram-negative bacterial members, such as Acidobacteria, Bacteroidetes, and Proteobacteria, also participated in the decomposition of callus under anoxic soil conditions. Our study showed that carbon flow into the diverse bacterial members during the callus decomposition and the distinctiveness of the bacterial communities was formed under the anoxic and oxic soil conditions. [ABSTRACT FROM AUTHOR]

The role of 1-pyrroline was studied via feeding experiments using rice calli cultures to gain further insight into the key steps of 2-acetyl-1-pyrroline (2AP) biosynthesis in rice. The origin of the acetyl donor was also studied through stable isotope labelled substrates. Incubation of fresh calli from a fragrant rice variety (Aychade) and a non-fragrant variety (Gladio × Fidji K2) with 1-pyrroline led to a significant increase in 2AP in both varieties. Importantly, the amount of 2AP in the non-fragrant variety could be greatly enhanced by this supplementation. When rice calli were fed with increasing levels of 1-pyrroline, 2AP levels increased accordingly. Our data show that 1-pyrroline is a limiting factor for 2AP synthesis in rice. Heat treatment of calli suggested that 1-pyrroline might be enzymatically acetylated. The presence of labelled 2AP in calli supplemented with [U- 13 C]glucose, sodium acetate (1,2- 13 C 2 ) and sodium octanoate (1,2,3,4- 13 C 4 ) suggested that these compounds are possible candidates for acetyl group-donors of 2AP, predominately in the form of intact labelled 13 C 2 -units. [ABSTRACT FROM AUTHOR]

We improved the aluminum borate whisker-mediated method of DNA delivery into rice callus (Oryza sativa L., cv. Notohikari). The following factors were examined: amount of whiskers, the kind of apparatus for agitation, the type of whiskers, duration of agitation, and agitation speed. Twenty callused scutellar tissues were agitated in a 1.5 ml microtube containing aluminum borate whiskers (ABW), liquid medium, and the plasmid pAct1-F carrying the β-glucuronidase (GUS) gene. After the agitation, the scutellar tissues were subjected to transient GUS expression assay, which visualizes GUS-expressing cells as blue spots. Efficiencies of DNA delivery were evaluated as the number of blue spots resulting from the assay. In the present study, we succeeded in improving the efficiency of DNA delivery by changing the apparatus for agitation from rotary (Vortex Genie 2) to multidirectional (MT-360), and the type of ABW from Alborex Y to an aminosilane-coated Alborex (Alborex YS3A).

Two distinctive structures were observed on the surface of rice callus treated with cell wall degrading enzymes, pectinase and cellulase by scanning electron microscopy; i.e., membranous, and the fibrillar structures. The membranous layer was digestible with the enzymes, while the fibrillar structures were not. The membranous matrix was mucilaginous, and positive to PAS reaction. Protoplasts were released after the enzyme treatment from the areas covered with the enzyme-digestible mucilaginous layer. The role of the extracellular matrix of the rice callus is discussed from the standpoint of growth and differentiation of the rice callus.

Purpose: Nitrification is a key process in the global nitrogen cycle, of which the first and rate-limiting step is catalyzed by ammonia monooxygenase. Root cap cells are one of substrates for microorganisms that thrive in the rhizosphere. The degradation of root cap cells brings about nitrification following ammonification of organic nitrogen derived from the root cap cells. This study was designed to gain insights into the response of ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) to mineralized N from root cap cells and the composition of active bacterial and archaeal ammonia oxidizers in rice soil. Materials and methods: Rice callus cells were used as a model for root cap cells, and unlabelled (C) and C-labelled callus cells were allowed to decompose in aerobic soil microcosms. Real-time quantitative polymerase chain reaction (PCR), DNA-based stable isotope probing (SIP), and denaturing gradient gel electrophoresis (DGGE) were applied to determine the copy number of bacterial and archaeal amoA genes and the composition of active AOB and AOA. Results and discussion: The growth of AOB was significantly stimulated by the addition of callus cells compared with the growth of AOA with a much lesser extent. AOB communities assimilated C derived from the callus cells, whereas no AOA communities grew on C-callus. Sequencing of the DGGE bands in the SIP experiments revealed that the AOB communities belonging to Nitrosospira spp. dominated microbial ammonia oxidation with rice callus amendment in soil. Conclusions: The present study suggests that root cap cells of rice significantly stimulated the growth of AOB, and the active members dominating microbial ammonia oxidation belonged to Nitrosospira spp. in rice rhizosphere. [ABSTRACT FROM AUTHOR]

Abstract: A rapid and reproducible method to develop transgenic plants with enhanced transformation efficiency using Agrobacterium has been developed for the elite indica rice variety BPT 5204. Different rice calli aged from 3 to 30 d were co-cultivated with pre-incubated Agrobacterium suspension culture (LBA4404: pSB1, pCAMBIA1301) and incubated in dark for 3 d. Based on the transient GUS gene expression analysis, 6-day-old young calli showed high transformation frequency followed by 21-day-old ones. Thus, both 6- and 21-day-old calli were used for assessing the stable transformation efficiency. It was observed that the 6-day-old young transformed calli showed about 2-fold higher regeneration frequency when compared with 21-day-old calli. The transformation efficiency was enhanced for young calli to 5.9% compared with 0.8% of the 21-day-old calli. Molecular and genetic analysis of transgenic plants (T0) revealed the presence of 1–2 copies of T-DNA integration in transformants and it follows Mendalian ratio in T1 transgenic plants. From the present study, it was concluded that the development of transgenic rice plants in less duration with high regeneration and transformation efficiency was achieved in BPT 5204 by using 6-day-old young calli as explants. [Copyright &y& Elsevier]

Highlights: [•] We expressed a recombinant major capsid protein (rMCP) of rock bream iridovirus in rice callus. [•] Fish immunized orally with rMCP underwent successful induction of antibodies and were protected against viral challenge. [•] Transgenic single-cell cultures offer advantages over whole-plant systems with high level containment. [•] Plant-based oral administration protocol could be used as an effective (less labor and stress) means of accomplishing practical vaccine trials. [Copyright &y& Elsevier]

Key message: We showed that rice prolamin polypeptides formed ER-derived PBs in transgenic rice calli, and that this heterologous transgene expression system is suitable for studying the mechanism of rice PB-I formation. Abstract: Rice prolamins, alcohol-soluble seed storage proteins, accumulate directly within the rough endoplasmic reticulum (ER) lumen, leading to the formation of ER-derived type I protein bodies (PB-Is) in rice seed. Because rice prolamins do not possess a well-known ER retention signal such as K(H)DEL, or a unique sequence for retention in the ER such as a tandem repeat domain of maize and wheat prolamins, the mechanisms of prolamin accumulation in the ER and PB-I formation are poorly understood. In this study, we examined the formation mechanisms of PBs by expressing four types of rice prolamin species fused to green fluorescent protein (GFP) in transgenic rice calli. Each prolamin-GFP fusion protein was stably accumulated in rice calli and formed ER-derived PBs. In contrast, GFP fused with the signal peptide of prolamin was secreted into the intercellular space in rice calli. In addition, each of the four types of prolamin-GFP fusion proteins was co-localized with the ER chaperone binding protein. These results suggest that the mature polypeptide of prolamin is capable of being retained in the ER and induce the formation of PBs in non-seed tissue, and that the rice callus heterologous transgene expression system is useful for studying the mechanisms of rice PB-I formation. [ABSTRACT FROM AUTHOR]

The activity of indole-3-acetamide (IAM) hydrolase from rice cells was enriched ca. 628-fold by gel filtration and anion exchange column chromatography. The molecular masses of the IAM hydrolase estimated by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis were approximately 50.5 kD and 50.0 kD, respectively. The enzyme exhibited maximum activity at pH 6.0–6.5. The enzyme was stable against heat treatments between 4 and 50°C and works optimally at 52°C. The activity remained constant at 4°C for at least 143 days. The purified enzyme fraction hydrolyzed indoleacetic acid ethyl ester (Et-IAA) in addition to IAM and its homologue, 1-naphthalene-acetamide, but not indole-3-acetonitrile. Km values of the enzyme were 0.96 mM and 0.55 mM for IAM and Et-IAA, respectively. Although the molecular mass of the enzyme was very similar to that of IAM hydrolase of Agrobacterium tumefaciens involved in tumor formation, the biochemical properties of the enzyme including its high Km value were considerably different from those of the A. tumefaciens enzyme. Based on these enzyme properties, we will discuss whether the amidohydrolase is involved in auxin biosynthesis in rice cells. [ABSTRACT FROM AUTHOR]

Ultrasound acts as an alternative stress on cells or tissues. In this study it is aimed to investigate the effect of ultrasonic stimulation on the growth and proliferation of Oryza sativa Nipponbare cells (rice callus) in suspension culture. After the samples were stimulated by ultrasound at 28 kHz, we measured their growth and proliferation by using a colorimetric MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay as well as fresh weights of the cells. Growth curves were obtained by fresh weights in the suspension culture after ultrasonic agitation for different time from 2 to 120 s. In MTT method, the optical density was determined at 570 nm in the cell suspension on 10 days after the ultrasonic agitation. Up to 5 s agitation OD570 increased; it decreased for more prolonged stimulation. We found that ultrasonic stimulation could promote the growth and proliferation of O. sativa Nipponbare cells in suspension culture with the optimal stimulation of 5 s, while with longer agitation, its growth and proliferation was inhibited. The mechanism may be that the ultrasound activated or destroyed the cellular structure, such as cell membrane, cytoskeleton and mitochondria in which many enzymes and ion channels are affected. In addition, the enhancement of cell wall and cell membrane fluidity might be one of the factors to promote the cell growth in 5-s ultrasonic stimulation. [Copyright &y& Elsevier]

A method was developed to maintain plant regeneration activity of rice cells ( Oryza sativa L.) using embryogenic callus. Calluses were cultured in suspension, then on solid medium, to form compact globular callus resistant to low-temperature stress and with high plant regeneration activity. Callus preserved at 5 °C for 5 months regenerated plants from protoplasts at a frequency higher than from non-preserved callus from cv. Nipponbare, and cv. Koshihikari, but at lower rates from cv. Akitakomachi. Similar results were obtained from protoplasts of the three cultivars. Callus preserved at 5 °C for 8 months incurred cell damage, yet some surviving cells divided in suspension culture and eventually regenerated whole plants. Preserved and non-preserved regenerated plants showed similar levels of somaclonal variation. [ABSTRACT FROM AUTHOR]

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR11) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD50 concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na+, K+, Cl−, Mg+2, and Ca+2 contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K+, Mg+2 and Ca+2. The accumulation of Na+ and Cl− ions was less in the hydroxyproline resistant calli. [ABSTRACT FROM AUTHOR]

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR11) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD50 concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth, proline content and activity of proline and IAA oxidases of the cultured tissues were determined at the end of 0, 2, 4, and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing hydroxyproline and NaCl showed increased dry weight and proline content as compared to NaCl stressed calli. The levels of proline and IAA oxidases were also low in the hydroxyproline resistant calli. [ABSTRACT FROM AUTHOR]

Background: β-carotene (provitamin A) is an important target for biofortification of crops as a potential solution to the problem of vitamin A deficiency that is prevalent in developing countries. A previous report showed that dominant expression of splicing variants in the Orange (Or) gene causes β-carotene accumulation in cauliflower curd. In this study, we focused on a putative orthologue of the cauliflower or gene in rice, Osor, and attempt to accumulate β-carotene in rice callus by modification of the Osor gene via genome editing using CRISPR/Cas9. Findings: CRISPR/Cas9 vectors for the Osor gene were constructed and transformed into rice calli. Some transformed calli showed orange color due to β-carotene hyper-accumulation. Molecular analyses suggest that orange-colored calli are due to an abundance of in-frame aberrant Osor transcripts, whereas out-of-frame mutations were not associated with orange color. Conclusions: We demonstrate that directed gene modification of the Osor gene via CRISPR/Cas9-mediated genome editing results in β-carotene fortification in rice calli. To date, golden rice, which accumulates β-carotene in rice endosperm, has been developed by conventional transgenic approaches. Our results suggest an alternative approach to enhancing β-carotene accumulation in crops. [ABSTRACT FROM AUTHOR]