Optimized medium composition in Physalis alkekengi callus culture altered nitric oxide level for inducing antioxidant enzyme activities and secondary metabolites.

Physalis alkekengi L. is a valuable medicinal plant from the Solanaceae family and has multiple therapeutic applications. This study aimed to develop an optimized protocol for callogenesis in P. alkekengi to obtain friable calluses with high biomass. The effect of different concentrations of picloram, casein hydrolysate (CH), basal media (Murashige and Skoog (MS) and Gamborg (B5)), and static magnetic field (SMF) were investigated on the callus induction and growth, signaling molecules, and enzymatic and non-enzymatic antioxidants. Results showed that CH (200 mgL-1) and SMF4 mT for 90 min increased callus induction and fresh weight in P. alkekengi, while different concentrations of picloram reduced callogenesis. Hypocotyl explants showed various callogenesis and metabolic responses depending on the basal medium type. The 2B5 medium supplied with CH 200 (mgL-1) induced friable and cream calluses with high biomass (0.62 g) compared to the MS medium (control). The maximum activity of superoxide dismutase and catalase activities was identified in the 2B5 medium and peroxidase in the 2MS medium. The highest total phenolic (129.44 µg g-1DW) content and phenylalanine-ammonia lyase activity were obtained in the 2MS medium, and total withanolides (49.86 µg g-1DW) and DPPH radical scavenging activity were observed in the 2B5 medium. The 2MS medium boosted the hydrogen peroxide and nitric oxide levels, while their contents alleviated in the 2B5 medium, although these parameters were higher than the control. The findings of this study suggest that an effective protocol for successful callogenesis in P. alkekengi and the nutrient composition of culture medium by affecting the level of signaling molecules can control the antioxidant defense system and callus growth.

A single-cell transcriptome atlas reveals the trajectory of early cell fate transition during callus induction in Arabidopsis.

The acquisition of pluripotent callus from somatic cells plays an important role in plant development studies and crop genetic improvement. This developmental process incorporates a series of cell fate transitions and reprogramming. However, our understanding of cell heterogeneity and mechanisms of cell fate transition during callus induction remains quite limited. Here, we report a time-series single-cell transcriptome experiment on Arabidopsis root explants that were induced in callus induction medium for 0, 1, and 4 days, and the construction of a detailed single-cell transcriptional atlas of the callus induction process. We identify the cell types responsible for initiating the early callus: lateral root primordium-initiating (LRPI)-like cells and quiescent center (QC)-like cells. LRPI-like cells are derived from xylem pole pericycle cells and are similar to lateral root primordia. We delineate the developmental trajectory of the dedifferentiation of LRPI-like cells into QC-like cells. QC-like cells are undifferentiated pluripotent acquired cells that appear in the early stages of callus formation and play a critical role in later callus development and organ regeneration. We also identify the transcription factors that regulate QC-like cells and the gene expression signatures that are related to cell fate decisions. Overall, our cell-lineage transcriptome atlas for callus induction provides a distinct perspective on cell fate transitions during callus formation, significantly improving our understanding of callus formation.

Efficient isolated microspore culture protocol for callus induction and plantlet regeneration in japonica rice (Oryza sativa L.).

BACKGROUND: Isolated microspore culture is a useful biotechnological technique applied in modern plant breeding programs as it can produce doubled haploid (DH) plants and accelerate the development of new varieties. Furthermore, as a single-cell culture technique, the isolated microspore culture provides an excellent platform for studying microspore embryogenesis. However, the reports on isolated microspore culture are rather limited in rice due to the low callus induction rate, poor regeneration capability, and high genotypic dependency. The present study developed an effective isolated microspore culture protocol for high-frequency androgenesis in four japonica rice genotypes. Several factors affecting the isolated microspore culture were studied to evaluate their effects on callus induction and plantlet regeneration. RESULTS: Low-temperature pre-treatment at 4 â„ƒ for 10-15 days could effectively promote microspore embryogenesis in japonica rice. A simple and efficient method was proposed for identifying the microspore developmental stage. The anthers in yellow-green florets located on the second type of primary branch on the rice panicle were found to be the optimal stage for isolated microspore culture. The most effective induction media for callus induction were IM2 and IM3, depending on the genotype. The optimal concentration of 2, 4-D in the medium for callus induction was 1 mg/L. Callus induction was negatively affected by a high concentration of KT over 1.5 mg/L. The differentiation medium suitable for japonica rice microspore callus comprised 1/2 MS, 2 mg/L 6-BA, 0.5 mg/L NAA, 30 g/L sucrose, and 6 g/L agar. The regeneration frequency of the four genotypes ranged from 61-211 green plantlets per 100 mg calli, with Chongxiangjing showing the highest regeneration frequency. CONCLUSIONS: This study presented an efficient protocol for improved callus induction and green plantlet regeneration in japonica rice via isolated microspore culture, which could provide valuable support for rice breeding and genetic research.

GWAS determined genetic loci associated with callus induction in oil palm tissue culture.

KEY MESSAGE: GWAS identified six loci at 25 kb downstream of WAK2, a crucial gene for cell wall and callus formation, enabling development of a SNP marker for enhanced callus induction potential. Efficient callus induction is vital for successful oil palm tissue culture, yet identifying genomic loci and markers for early detection of genotypes with high potential of callus induction remains unclear. In this study, immature male inflorescences from 198 oil palm accessions (dura, tenera and pisifera) were used as explants for tissue culture. Callus induction rates were collected at one-, two- and three-months after inoculation (C1, C2 and C3) as phenotypes. Resequencing generated 11,475,258 high quality single nucleotide polymorphisms (SNPs) as genotypes. GWAS was then performed, and correlation analysis revealed a positive association of C1 with both C2 (R = 0.81) and C3 (R = 0.50), indicating that C1 could be used as the major phenotype for callus induction rate. Therefore, only significant SNPs (P ≤ 0.05) in C1 were identified to develop markers for screening individuals with high potential of callus induction. Among 21 significant SNPs in C1, LD block analysis revealed six SNPs on chromosome 12 (Chr12) potentially linked to callus formation. Subsequently, 13 SNP markers were identified from these loci and electrophoresis results showed that marker C-12 at locus Chr12_12704856 can be used effectively to distinguish the GG allele, which showed the highest probability (69%) of callus induction. Furthermore, a rapid SNP variant detection method without electrophoresis was established via qPCR-based melting curve analysis. Our findings facilitated marker-assisted selection for specific palms with high potential of callus induction using immature male inflorescence as explant, aiding ortet palm selection in oil palm tissue culture.

An Efficient Agrobacterium-Mediated Genetic Transformation System for Gene Editing in Strawberry (Fragaria × ananassa).

The octoploid-cultivated strawberry variety Benihope (Fragaria × ananassa Duch cv. Benihope) is an important commercial plant. It is highly susceptible to different diseases, which ultimately leads to a reduction in yield. Gene-editing methods, such as CRISPR/Cas9, demonstrate potential for improving disease resistance in the strawberry cv. Benihope. Establishing a plant regeneration system suitable for CRISPR/Cas9 gene editing is crucial for obtaining transgenic plants on a large scale. This research established a callus induction and plant regeneration system for Agrobacterium-mediated CRISPR/Cas9 gene editing in strawberry cv. Benihope by evaluating multiple types of explants and various plant growth regulators throughout the entire tissue culture process. The results showed that the efficiency of callus induction is strongly influenced by the type of explant and is highly sensitive to the combination of plant growth regulators. Among the different plant growth regulators employed, thidiazuron (TDZ), in combination with 2,4-dichlorophenoxyacetic acid (2,4-D), effectively induced callus formation and plant regeneration from explants derived from nutrient tissues such as runner tips and crowns. In addition, the regeneration experiment demonstrated that the addition of polyvinylpyrrolidone (PVPP) to the shoot regeneration medium could inhibit tissue browning. The gene-edited plants in which some or all of the Fvb7-1, Fvb7-2, Fvb7-3, and Fvb7-4 genes in the MLO (Mildew resistance Locus O) gene family were knocked out by CRISPR/Cas9 system were obtained by applying the plant regeneration system developed in this study.

Plant regeneration from embryogenic callus-derived from immature leaves of Momordica charantia L.

Bitter melon (Momordica charantia L.), a widely cultivated food and medicinal plant native to the world's subtropics and tropics, is a Cucurbitaceae rich in carotenoids. However, the low seed germination frequency and progeny variability associated with the production of this plant have a substantial impact on its growth and yield. These constraints affect the availability and exploitation of this crop, especially the fruits, which are rich in secondary metabolites such as ß-carotene and α-carotene. In vitro regeneration would help overcome the obstacle linked to the germination of this plant and increase its yield and utilization. A reproducible in vitro organogenesis protocol was established using bitter melon embryogenic callus derived from immature leaf explants of in vivo grown seedlings and in vitro plantlets. Regeneration via callus was conducted on MSB5 media augmented with different plant growth regulator concentrations. The maximum frequency of callus formation (95.09 %) was produced in MSB5 media incorporated with 1.2 mg L-1 NAA augmented with 0.5 mg L-1 TDZ. MSB5 medium with no growth regulators was observed to be the most suitable for the shoot and root formation from the callus, producing a significantly high shoot percentage of 90.91 % and 21.53 shoots per explants, and the highest rooting frequency and root number of 88.92 % and 6.23 roots per explant, respectively, from leaf-derived callus of in vitro plantlets. The elongated plantlets had grown to a significantly higher average height of 12.20 cm on media added with 0.75 mg L-1 GA3. This reproducible method for regenerating bitter melon plantlets could facilitate mass multiplication, conservation, and commercial field production.

A Versatile Protocol for Efficient Transformation and Regeneration in Mega Indica Rice Cultivar MTU1010: Optimization through Hormonal Variables.

Rice is one of the apex food crops in terms of meeting the daily calorific and dietary requirement of the majority of the world population. However, rice productivity is severely limited by various biotic and abiotic attributes, causing a severe threat to global food security. In the use of functional genomics and genome editing for the generation of trait-enhanced genotypes, it is necessary to have an efficient genetic transformation and regeneration protocol. The recalcitrant nature and paucity of efficient and versatile genetic transformation and regeneration protocols for indica cultivars remains a constraint. In the present study, we have optimized a tissue culture method for MTU1010, a mega indica rice variety. We conducted a combinatorial analysis of different plant growth regulators on embryogenic callus induction efficiency, and it was observed that MSB5 medium supplemented with 2.5 mg/L 2-4D and 0.25 mg/L 6-BAP results in maximum embryogenic callus induction, i.e., 92%. The regeneration efficiency of a transformed callus can be enhanced by up to 50% with the supplementation of 1 mg/L kinetin alongside 2.5 mg/L BAP and 0.5 mg/L NAA in the shooting medium. Furthermore, our results unveiled that the pre-activation of Agrobacterium culture for 30 min with 150 µM acetosyringone significantly increased the transformation efficiency of calli. Additionally, descaling the salt concentration to half strength in resuspension and co-cultivation increased the efficiency of transformation up to 33%. Thus, the protocol developed in this study will be instrumental for the genome editing and genetic engineering of indica rice cultivars for functional genomics studies and crop improvement.

Production of Dihydroisocoumarins by Callus Induction from Hydrangea macrophylla var. thunbergii Leaves.

Dihydroisocoumarins, hydrangenol 8-O-ß-D-glucopyranoside (1), phyllodulcin 8-O-ß-D-glucopyranoside (2), hydrangenol (3), and phyllodulcin (4), are well-known as the major secondary metabolites in the leaves of Hydrangea macrophylla var. thunbergii. Dihydroisocoumarins are pharmaceutical compounds with diverse bioactivity. Although dihydroisocoumarins are commonly isolated from Hydrangea plants or via organic chemical synthesis, their production via callus induction is considered a promising alternative. In the present study, callus induction and proliferation of H. macrophylla var. thunbergii, and constituents 1-4 were quantified in calluses cultured in 17 different media. We found that the combination of the phytohormones 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA) was useful for callus proliferation in H. macrophylla var. thunbergii. The balance and concentrations of indole-3-acetic acid (IAA) and BA greatly affected the contents of 1-4. Particularly, 1 (2.03-3.46% yield from the dry callus) was successfully produced from the callus induced by IAA (0.5 mg/L) and BA (1.0 mg/L) at yields comparable to isolated yields from plants. To the best of our knowledge, this is the first study to show that the calluses of H. macrophylla var. thunbergii contained 1. These findings may be useful for producing bioactive dihydroisocoumarins.

Fungal Elicitation Enhances Vincristine and Vinblastine Yield in the Embryogenic Tissues of Catharanthus roseus.

Fungal elicitation could improve the secondary metabolite contents of in vitro cultures. Herein, we report the effect of Fusarium oxysporum on vinblastine and vincristine alkaloid yields in Catharanthus roseus embryos. The study revealed increased yields of vinblastine and vincristine in Catharanthus tissues. Different concentrations, i.e., 0.05% (T1), 0.15% (T2), 0.25% (T3), and 0.35% (T4), of an F. oxysporum extract were applied to a solid MS medium in addition to a control (T0). Embryogenic calli were formed from the hypocotyl explants of germinating seedlings, and the tissues were exposed to Fusarium extract elicitation. The administration of the F. oxysporum extract improved the growth of the callus biomass, which later differentiated into embryos, and the maximum induction of somatic embryos was noted T2 concentration (102.69/callus mass). A biochemical analysis revealed extra accumulations of sugar, protein, and proline in the fungus-elicitated cultivating tissues. The somatic embryos germinated into plantlets on full-strength MS medium supplemented with 2.24 µM of BA. The germination rate of the embryos and the shoot and root lengths of the embryos were high at low doses of the Fusarium treatment. The yields of vinblastine and vincristine were measured in different treated tissues via high-pressure thin-layer chromatography (HPTLC). The yield of vinblastine was high in mature (45-day old) embryos (1.229 µg g-1 dry weight), which were further enriched (1.267 µg g-1 dry weight) via the F. oxysporum-elicitated treatment, especially at the T2 concentration. Compared to vinblastine, the vincristine content was low, with a maximum of 0.307 µg g-1 dry weight following the addition of the F. oxysporum treatment. The highest and increased yields of vinblastine and vincristine, 7.88 and 15.50%, were noted in F. oxysporum-amended tissues. The maturated and germinating somatic embryos had high levels of SOD activity, and upon the addition of the fungal extracts, the enzyme's activity was further elevated, indicating that the tissues experienced cellular stress which yielded increased levels of vinblastine and vincristine following the T2/T1 treatments. The improvement in the yields of these alkaloids could augment cancer healthcare treatments, making them easy, accessible, and inexpensive.

Shoot Organogenesis and Regeneration from Leaf Seedlings of Diospyros oleifera Cheng.

Persimmons (Diospyros) are economically important trees that are widely cultivated for wood production in China, and Diospyros oleifera Cheng is the main persimmon grafting stock. However, an efficient tissue culture system has not been perfected for D. oleifera due to the limits of proliferation and rooting cultures. Therefore, this study examined the effects of different plant growth regulators and concentrations on the primary culture of young embryos, induction of leaf callus, differentiation of adventitious shoots, and rooting culture of D. oleifera. The optimal formula for young embryo germination was 1/2 Murashige and Skoog (MS) medium containing 0.5 mg/L gibberellic acid (GA3); after 25 days, the sprouting rate of the young embryos was 67.3%. The best medium for leaf callus induction was 1/2MS medium containing 2.0 mg/L of 6-benzylaminopurine (6-BA) and 0.5 mg/L of naphthaleneacetic acid (NAA), and the callus induction rate was 88.9%. Then, the callus was transferred to 1/2MS medium containing 2.0 mg/L of zeatin (ZT), 0.5 mg/L of NAA, and 2.0 mg/L of thidiazuron (TDZ) to induce adventitious shoots; after 25 days, 5.4 buds were produced per explant, and the induction rate of the adventitious shoots was 88.3%. The adventitious shoots were transferred to 1/2MS medium containing 2.0 mg/L of ZT, 2.0 mg/L of 6-(γ,γ-dimethylallylamino)purine (2iP), and 0.1 mg/L of indole acetic acid (IAA) for the proliferation culture, for which the multiplication coefficient approached 7.5. After multiplication, the adventitious shoots were inoculated into 1/2MS medium containing 1.0 mg/L of indole butyric acid (IBA), 0.5 mg/L of NAA, and 1.0 mg/L of kinetin (KT); the rooting rate was 60.2%, and the average number of roots was 6.9.

An Efficient Transformation System for Fast Production of VcCHS Transgenic Blueberry Callus and Its Expressional Analysis.

The Agrobacterium tumefaciens-mediated transformation for blueberries remains less efficient than is desirable. A new leaf callus regeneration and genetic transformation system was investigated in blueberries in this study. The leaf explants of cv. 'Legacy' and 'Northland' were used to establish the stable callus induction system when placed on the woody plant medium (WPM) supplemented with 1.0 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA for 30 d; then, the callus was sub-cultured in the proliferation medium supplemented with 1.5 mg·L-1 2, 4-D, 0.4 mg·L-1 6-BA in the darkness at 25 °C every 30 days. The co-cultivation of callus with A. tumefaciens was operated on WPM plus 100 µM acetosyringone for 4 days; then, the transferred callus was grown in WPM supplemented with 1.5 mg·L-1 2,4-D, 0.4 mg·L-1 6-BA, 50 mg·L-1 hygromycin, and 200 mg·L-1 cefotaxime. The VcCHS transgenic blueberry callus with both GFP signal and Hyg resistance was obtained from the transformed callus of cv. 'Northland'. The rate of GFP signal detected in the transformed callus was as high as 49.02%, which was consistent with the PCR assay. Collectively, this study provides a highly efficient genetic transformation system in blueberry callus and a powerful approach for the molecular breeding of blueberries.

Callus Derived from Petals of the Rosa hybrida Breeding Line 15R-12-2 as New Material Useful for Fragrance Production.

Rose (Rosa hybrida) is a major flower crop worldwide and has long been loved for its variety of colors and scents. Roses are mainly used for gardening or cutting flowers and are also used as raw materials for perfumes, cosmetics, and food. Essential oils, which are extracted from the flowers of plants, including roses, have various scents, and the essential oil market has been growing steadily owing to the growing awareness of the benefits of natural and organic products. Therefore, it is necessary to develop a system that stably supplies raw materials with uniform ingredients in line with the continuous increase in demand. In this study, conditions for the efficient induction of callus were established from the petals of the rose breeding line 15R-12-2, which has a strong scent developed by the National Institute of Horticultural and Herbal Science, Rural Development Administration. The highest callus induction rate (65%) was observed when the petals of the fully open flower (FOF) were placed on the SH11DP medium so that the abaxial surface was in contact with the medium. In addition, the VOCs contained in the petals of 15R-12-2 and the petal-derived callus were analyzed by HS-SPME-GC-MS. Thirty components, including esters and alcohols, were detected in the petal-derived callus. Among them, 2-ethylhexan-1-ol, which showed 59.01% relative content when extracted with hexane as a solvent, was the same component as detected in petals. Therefore, petal-derived callus is expected to be of high industrial value and can be suggested as an alternative pathway to obtaining VOCs.

Agrobacterium-mediated Genetic Transformation of Cotton and Regeneration via Somatic Embryogenesis.

Cotton is a significant industrial crop, playing an essential role in the global economy that suffers several setbacks due to biotic and abiotic adversities. Despite such problems, biotechnological advances in cotton are limited because of genetic transformation and regeneration limitations. Here, we present a detailed protocol optimized based on previously published papers, along with our modifications. These involve changes in Agrobacterium concentration, co-cultivation time and temperature, hormones used for regeneration, media manipulation for embryogenic callus production, and efficient rescue of deformed embryos. Further, this protocol has been used in genetic studies on biotic and abiotic stress in cotton. This protocol assures a reproducible stable transgenic cotton development procedure via somatic embryogenesis that can be used by researchers worldwide. This protocol was validated in: Nat Biotechnol (2016), DOI: 10.1038/nbt.3665.

Shoot differentiation from Dendrocalamus brandisii callus and the related physiological roles of sugar and hormones during shoot differentiation.

Only a few calli regeneration systems of bamboos were successfully established, which limited the research on the physiological mechanism of callus differentiation. In this study, we successfully established the callus differentiation systems of Dendrocalamus brandisii (Munro) via seeds. The results showed that the best medium for the callus induction of D. brandisii seeds was basal Murashige and Skoog (1962) (MS) media amended with 5.0 mg l-1 2,4-D and 0.5 mg l-1 kinetin (KT), and the optimal medium for shoot differentiation was the basal MS media supplemented with 4.0 mg l-1 6-benzylaminopurine (6-BA) and 0.5 mg l-1 1-Naphthaleneacetic acid (NAA). Callus tissues had apparent polarity in cell arrangement and developed their own meristematic cell layers. Alpha-amylase (α-amylase), starch phosphorylase (STP) and sucrose synthase (SUSY) played a dominant role in carbohydrate degradation in callus during shoot differentiation. The pentose phosphate pathway (PPP) and TCA pathways were up-regulated in the shoot-differentiated calli. The dynamics of 6-BA and KT contents in calli were consistent with their concentrations applied in medium. Indoleacetic acid (IAA) synthesis and the related signal transduction were down-regulated, whereas the endogenous CTK contents were up-regulated by the exogenous cytokinin (CTK) application in shoot-differentiated calli, and their related synthesis, transport and signal transduction pathways were also up-regulated. The down-regulated signal transduction pathways of IAA and abscisic acid (ABA) revealed that they did not play the key role in the shoot differentiation of bamboos. Gibberellins (GAs) also played a role in shoot differentiation based on the down-regulation of DELLA and the up-regulation of PIF4 genes. The overexpression of DbSNRK2 and DbFIF4 genes further confirmed the negative role of ABA and the positive role of GAs in shoot differentiation.

Plant regeneration via callus-mediated organogenesis in commercial variety of Chuanbeichai No. 1 in Bupleurum chinense DC.

Bupleurum chinense DC is an important medicinal plant with many active ingredients that are used for the treatment of different types of diseases and valued in pharmaceutical markets. In vitro shoot regeneration can efficiently contribute to the improvement of B. chinense. In the present study, we investigated the effects of the explant type and plant growth regulators (PGRs) on embryogenic callus induction and plant regeneration in B. chinense. Our investigation demonstrated that 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 1 mg/L thidiazuron (TDZ) played a major role in promoting callus induction from leaf, hypocotyl and stem 2 explants, whereas the most effective treatment for stem 1 callus formation was Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kin). The highest shoot regeneration rate (57.14%) was obtained from hypocotyl-induced calli in MS medium with 0.5 mg/L Kin after 12 weeks of cultivation. This regeneration protocol can be used in large-scale cultivation and may be useful for future genetic modifications of B. chinense .

Generation of doubled haploids in cauliflower.

Hybrids of cauliflower are in high demand world over due to their high yield potential, earliness, better quality, better resistance to biotic and abiotic stresses. Conventionally, hybrids are developed from the intercrossing of two diverse inbred parental lines which are developed through continuous inbreeding for 8-10 generations and still don't attain complete homozygosity. Doubled haploid technology on the other hand generate completely homozygous inbred lines in a single step. Therefore, a study was undertaken at Punjab Agricultural University, Ludhiana, to develop a protocol for the development of doubled haploid lines in cauliflower. The anthers were excised from the flower buds of different genotypes viz. Jyoti, Pusa Sharad, Kartiki, CAUMH-2, CAUMH-10, LS-2, LS-3, and LS-5 followed by their culture on five different callus induction media compositions. Genotypes differed significantly in the ability to induce callus which was maximum in Jyoti followed by LS-2. Different media compositions also varied significantly in callus induction efficiency which was maximum on MS media+1.5 mg/L 2,4-D +1.0 mg/L NAA. Maximum shoot regeneration was recorded in genotype Kartiki followed by LS-2 when cultured on MS media+3.0 mg/L BAP+2.0 mg/L Kin. The regenerated shoots thus obtained were rooted on ½ MS media +1.0 mg/L IBA. Ploidy analysis of root tips revealed that 22.2% of the regenerated plantlets were haploids, 27.8% were spontaneous doubled haploids, 16.7% were tetraploids and remaining 33.3% were mixoploids.

Callus induction and transcriptomic analysis of in vitro embryos at different developmental stages of peony.

The efficient induction of peony embryogenic callus is of great significance to the improvement and establishment of its regeneration technology system. In this study, the in vitro embryos of 'Fengdanbai' at different developmental stages were selected as explants, the effects of different concentrations and types of plant growth regulator combinations on the induction and proliferation of embryonic callus at different developmental stages were investigated, and comparative transcriptome analysis of callus with different differentiation potentials were performed to explore the molecular mechanisms affecting callus differentiation. The results showed that the germination rate of 90d seed embryo was the best, which was 94.17%; the 70d and 80d cotyledon callus induction effect was the best, both reaching 100%, but the 80d callus proliferation rate was higher, the proliferation rate reached 5.31, and the optimal induction medium was MS+0.1 mg·L-1NAA+0.3 mg·L-1TDZ+3 mg·L-12,4-D, the callus proliferation multiple was 4.77. Based on the comparative transcriptomic analysis, we identified 3470 differentially expressed genes (DEGs) in the callus with high differentiation rate and low differentiation rate, including 1767 up-regulated genes and 1703 down-regulated genes. Pathway enrichment analysis showed that the "Phenylpropanoid biosynthesis" metabolic pathway was significantly enriched, which is associated with promoting further development of callus shoots and roots. This study can provide reference for genetic improvement and the improvement of regeneration technology system of peony.

Evaluation of the Anti-Cancer Potential of Rosa damascena Mill. Callus Extracts against the Human Colorectal Adenocarcinoma Cell Line.

Chemotherapy is an aggressive form of chemical drug therapy aiming to destroy cancer cells. Adjuvant therapy may reduce hazards of chemotherapy and help in destroying these cells when obtained from natural products, such as medical plants. In this study, the potential therapeutic effect of Rosa damascena callus crude extract produced in vitamin-enhanced media is investigated on colorectal cancer cell line Caco-2. Two elicitors, i.e., L-ascorbic acid and citric acid at a concentration of 0.5 g/L were added to the callus induction medium. Callus extraction and the GC-MS analysis of methanolic crude extracts were also determined. Cytotoxicity, clonogenicity, proliferation and migration of Caco-2 colorectal cancer cells were investigated using MTT cytotoxicity, colony-forming, Ki-67 flow cytometry proliferation and Migration Scratch assays, respectively. Our results indicated that L-ascorbic acid treatment enhanced callus growth parameters and improved secondary metabolite contents. It showed the least IC50 value of 137 ug/mL compared to 237 ug/mL and 180 ug/mL in the citric acid-treated and control group. We can conclude that R. damascena callus elicited by L-ascorbic acid improved growth and secondary metabolite contents as well as having an efficient antiproliferative, anti-clonogenic and anti-migratory effect on Caco-2 cancer cells, thus, can be used as an adjuvant anti-cancer therapy.

Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant.

SR4 genotype of rice is high altitude Himalayan rice prone to various abiotic stresses such as cold stress and therefore gives a poor yield. An efficient protocol for callusing and regeneration via direct and indirect means was established using mature seeds as an explant which can be utilized for molecular studies for genetic advancement of Himalayan rice genotype SR4 through transformation. Highest frequency (96.6%) of callus induction was obtained on MS media 3.0 mg/L 2, 4-D. While maximum regeneration frequency (100%), number of shoots with maximum length 9.14 ± 0.204 (cm) from callus was recovered from MS media amended with 5.0 mg/L BAP in combination with 0.5 mg/L NAA with highest number of shoots having an average shoot length 9.14 ± 0.204 (cm) after four weeks of culture. Direct multiple shoot regeneration from seed explants was obtained using various concentrations of TDZ and BAP with highest regeneration frequency was observed on MS media fortified with 6 mg/L of TDZ with maximum number of shoots. The shoots developed roots on MS media supplemented with 0.6 mg/L IBA.

Genome Estimation and Phytochemical Compound Identification in the Leaves and Callus of Abrus precatorius: A Locally Endangered Plant from the Flora of Saudi Arabia.

Abrus precatorius is considered to be a valuable source of natural products for the development of drugs against various diseases. Herein, the genome size and phytochemical compounds in the leaves and callus of A. precatorius were evaluated. The endangered A. precatorius was collected from the Al-Baha mountains, Saudi Arabia and identified based on the phylogenetic analysis of a DNA sequence amplified by ITS1 and ITS4 primers. The callus was induced by the culture of stem explants onto Murashige and Skoog medium (MS) supplemented with various combinations of 2,4-dichlorophenoxyacetic acid (2,4D) and 6-Benzylaminopurine (BAP). The callus with the highest fresh weight (2.03 g) was obtained in the medium containing 0.5µM BA and 5 µM 2,4-D after 8 weeks of culture; thus, the callus of this combination was selected for the genome estimation and phytochemical compound extraction. The genetic stability of the leaves from the donor as well as in the regenerated callus was analyzed by flow cytometry with optimized tomato (2C = 1.96 pg) as an external reference standard. The 2C DNA content was estimated to 1.810 pg ± 0.008 and 1.813 pg ± 0.004 for the leaves and callus, respectively. Then, the total phenol and total flavonoid contents in the methanol extract of the callus and leaves were measured using a spectrophotometer and the High-performance liquid chromatography (HPLC ) methods. The results showed that the methanolic extract of the leaves was higher in total phenols and total flavonoids than the callus extract. Finally, the extracts of callus and leaves were analyzed for phytochemical compound through the Gas chromatography and Mass spectroscopy (GC-MS). A total of 22 and 28 compounds were detected in the callus and leaves, respectively. The comparative analysis showed that 12 compounds of the secondary metabolites were present in both extracts.