Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don.

This study aimed to investigate the impact of plant growth regulators, sucrose concentration, and the number of subcultures on axillary shoot multiplication, in vitro flowering, and somaclonal variation and to assess the phytochemical composition, antioxidant capacity, and enzyme inhibitory potential of in vitro-established callus, somaclonal variant, and normal green shoots of Catharanthus roseus. The highest shoot induction rate (95.8%) and highest number of shoots (23.6), with a mean length of 4.5 cm, were attained when the C. roseus nodal explants (0.6-1 cm in length) were cultivated in Murashige and Skoog (MS) medium with 2 µM thidiazuron, 1 µM 2-(1-naphthyl) acetic acid (NAA), and 4% sucrose. The in vitro flowering of C. roseus was affected by sucrose, and the number of subcultures had a significant effect on shoot multiplication and somaclonal variation. The highest levels of phenolics and flavonoids were found in normal green shoots, followed by those in somaclonal variant shoots and callus. The phytochemicals in C. roseus extracts were qualified using liquid chromatography-tandem mass spectrometry. A total of 39, 55, and 59 compounds were identified in the callus, somaclonal variant shoot, and normal green shoot tissues, respectively. The normal green shoot extracts exhibited the best free radical scavenging ability and reducing power activity. The strongest acetylcholinesterase inhibitory effects were found in the callus, with an IC50 of 0.65 mg/mL.

Micropropagation and Quantification of Bioactive Compounds in Mertensia maritima (L.) Gray.

The goal of this study was to establish an efficient protocol for the large-scale propagation of Mertensia maritima (L.) Gray, and evaluate the carotenoid, fatty acid, and tocopherol contents in the leaves of in vitro regenerated shoots. Surface-disinfected node and shoot tip explants were placed on semisolid Murashige and Skoog (MS) medium with 0-16 µM N6-benzyladenine (BA), kinetin, (KN), and thidiazuron (TDZ) alone, or in combination with, 1 or 2 µM α-naphthaleneacetic acid (NAA). Of the three different cytokinins employed, TDZ elicited the best results for axillary shoot proliferation. A maximum frequency of shoot initiation above 84%, with a mean of 8.9 and 4.8 shoots per node and shoot tip, respectively, was achieved on the culture medium supplemented with 4 µM TDZ. A combination of TDZ + NAA significantly increased the percentage of multiple shoot formation and number of shoots per explant. The best shoot induction response occurred on MS medium with 4 µM TDZ and 1 µM NAA. On this medium, the node (93.8%) and shoot tip (95.9%) explants produced an average of 17.7 and 8.6 shoots, respectively. The highest root induction frequency (97.4%) and number of roots per shoot (25.4), as well as the greatest root length (4.2 cm), were obtained on half-strength MS medium supplemented with 4 µM indole-3-butyric acid (IBA). The presence of six carotenoids and α-tocopherol in the leaf tissues of M. maritima was confirmed by HPLC. Gas chromatography-mass spectrometry analysis confirmed the presence of 10 fatty acids, including γ-linolenic acid and stearidonic acid in the leaf tissues of M. maritima. All-E-lutein (18.49 µg g-1 fresh weight, FW), α-tocopherol (3.82 µg g-1 FW) and α-linolenic acid (30.37%) were found to be the significant compounds in M. maritima. For the first time, a successful protocol has been established for the mass propagation of M. maritima with promising prospects for harnessing its bioactive reserves.

In vitro propagation of Cymbidium goeringii Reichenbach fil. through direct adventitious shoot regeneration.

The influence of 2,4-dichlorophenoxyacetic acid (2,4-D), benzyladenine (BA), and thidiazuron (TDZ) on direct rhizome induction and shoot formation from rhizome explants of Cymbidium goeringii was explored. Rhizome segments obtained from in vitro seed cultures of C. goeringii were placed on Murashige and Skoog (MS) medium incorporated with 5, 10, 20, or 40 µM 2,4-D and 1, 2, 4, or 8 µM BA or TDZ alone or in combination with 20 µM 2,4-D. The explants developed only rhizomes on MS medium with or without 2,4-D. The highest percent of rhizome formation (100%) was obtained on MS medium incorporated with 20 µM of 2,4-D. The morphology and number of rhizomes varied with the level of 2,4-D in the medium. Direct adventitious shoot formation was achieved on medium incorporated with BA or TDZ. The adventitious shoots produced per explant significantly increased with the supplementation of 2,4-D to cytokinin-containing medium. The highest mean of 21.8 ± 1.8 shoot buds per rhizome segment was obtained in medium fortified with 20 µM 2,4-D and 2 µM TDZ. The greatest percent of root induction (100%) and the mean of 5.3 ± 1.1 roots per shoot were achieved on ½ MS medium incorporated with 2 µM of α-naphthaleneacetic acid. About 97% of the in vitro-produced plantlets acclimatized in the greenhouse. An efficient in vitro propagation protocol was thus developed for C. goeringii using rhizome explants.

Micropropagation of Ajuga species: a mini review.

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82-100% survival rate.

Development of S Haplotype-Specific Markers to Identify Genotypes of Self-Incompatibility in Radish (Raphanus sativus L.).

Radish (Raphanus sativus L.), a root vegetable belonging to the Brassicaceae family, is considered one of the representative crops displaying sporophytic self-incompatibility (SSI). The utilization of a self-incompatibility system in F1 breeding can improve the efficiency of cross-combinations, leading to a reduction in breeding time and aiding in the development of novel F1 varieties. The successful implementation of this system necessitates the rapid and accurate identification of S haplotypes in parental lines. In this study, we identified a total of nine S haplotypes among 22 elite radish lines through Sanger sequencing. Subsequently, we obtained sequences for showing a 95% similarity to nine S haplotypes, along with sequences identified by other researchers using BLAST. Following this, multiple sequence alignment (MSA) was conducted to identify SRK and SLG sequence similarities, as well as polymorphisms within the class I and II groups. Subsequently, S haplotype-specific marker sets were developed, targeting polymorphic regions of SRK and SLG alleles. These markers successfully amplified each of the nine S haplotypes. These markers will play a crucial role in the rapid and precise identification of parental S haplotypes in the radish F1 breeding process, proving instrumental in the radish F1 purity test.

VPBR: An Automatic and Low-Cost Vision-Based Biophysical Properties Recognition Pipeline for Pumpkin.

Pumpkins are a nutritious and globally enjoyed fruit for their rich and earthy flavor. The biophysical properties of pumpkins play an important role in determining their yield. However, manual in-field techniques for monitoring these properties can be time-consuming and labor-intensive. To address this, this research introduces a novel approach that feeds high-resolution pumpkin images to train a mathematical model to automate the measurement of each pumpkin's biophysical properties. Color correction was performed on the dataset using a color-checker panel to minimize the impact of varying light conditions on the RGB images. A segmentation model was then trained to effectively recognize two fundamental components of each pumpkin: the fruit and vine. Real-life measurements of various biophysical properties, including fruit length, fruit width, stem length, stem width and fruit peel color, were computed and compared with manual measurements. The experimental results on 10 different pumpkin samples revealed that the framework obtained a small average mean absolute percentage error (MAPE) of 2.5% compared to the manual method, highlighting the potential of this approach as a faster and more efficient alternative to conventional techniques for monitoring the biophysical properties of pumpkins.

Comparative analysis of RNA silencing suppression activities between viral suppressors and an endogenous plant RNA-dependent RNA polymerase.

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in eukaryotes, including higher plants. To counteract this, several plant viruses express silencing suppressors that inhibit RNA silencing in host plants. Here, we show that both 2b protein from peanut stunt virus (PSV) and a hairpin construct (designated hp-RDR6) that silences endogenous RNA-dependent RNA polymerase 6 (RDR6) strongly suppress RNA silencing. The Agrobacterium infiltration system was used to demonstrate that both PSV 2b and hp-RDR6 suppressed local RNA silencing as strongly as helper component (HC-Pro) from potato virus Y (PVY) and P19 from tomato bush stunt virus (TBSV). The 2b protein from PSV eliminated the small-interfering RNAs (siRNAs) associated with RNA silencing and prevented systemic silencing, similar to 2b protein from cucumber mosaic virus (CMV). On the other hand, hp-RDR6 suppressed RNA silencing by inhibiting the generation of secondary siRNAs. The small coat protein (SCP) of squash mosaic virus (SqMV) also displayed weak suppression activity of RNA silencing. Agrobacterium-mediated gene transfer was used to investigate whether viral silencing suppressors or hp-RDR6 enhanced accumulations of green fluorescence protein (GFP) and ß-glucuronidase (GUS) as markers of expression in leaf tissues of Nicotina benthamiana. Expression of both GFP and GUS was significantly enhanced in the presence of PSV 2b or CMV 2b, compared to no suppression or the weak SqMV SCP suppressor. Co-expression with hp-RDR6 also significantly increased the expression of GFP and GUS to levels similar to those induced by PVY HC-Pro and TBSV P19.

Seasonal Effects of Glucosinolate and Sugar Content Determine the Pungency of Small-Type (Altari) Radishes (Raphanus sativus L.).

Kimchi made from small-type (Altari) radishes grown in late spring is more pungent than that made from autumn-grown Altari radishes, which poses a major challenge in the kimchi industry. The mechanism through which the pungency of Altari radish changes seasonally has not been intensively investigated. In this study, three small-type radish cultivars with different pungency levels were cultivated in spring and autumn to identify the factors affecting the seasonal-dependent pungency of small-type radishes. The contents of pungency-related metabolite glucoraphasatin and other polar metabolites were analyzed. Although a previous study reported that the glucoraphasatin concentration affects the pungency of radish, in the current study, the concentration of neither glucoraphasatin nor its hydrolysis product (raphasatin) could fully explain the change in the pungency associated with radish cultivars grown in the two seasons. The change in the pungency of radish by season may be explained by the ratio of raphasatin content to total sweetness of sugars. In addition, the polar metabolites that differ with season were analyzed to identify seasonal biomarkers and understand the seasonal changed physio-biochemistry.

Genotyping-by-Sequencing-Based Genome-Wide Association Studies of Fusarium Wilt Resistance in Radishes (Raphanus sativus L.).

Fusarium wilt (FW) is a fungal disease that causes severe yield losses in radish production. The most effective method to control the FW is the development and use of resistant varieties in cultivation. The identification of marker loci linked to FW resistance are expected to facilitate the breeding of disease-resistant radishes. In the present study, we applied an integrated framework of genome-wide association studies (GWAS) using genotyping-by-sequencing (GBS) to identify FW resistance loci among a panel of 225 radish accessions, including 58 elite breeding lines. Phenotyping was conducted by manual inoculation of seedlings with the FW pathogen, and scoring for the disease index was conducted three weeks after inoculation during two constitutive years. The GWAS analysis identified 44 single nucleotide polymorphisms (SNPs) and twenty putative candidate genes that were significantly associated with FW resistance. In addition, a total of four QTLs were identified from F2 population derived from a FW resistant line and a susceptible line, one of which was co-located with the SNPs on chromosome 7, detected in GWAS study. These markers will be valuable for molecular breeding programs and marker-assisted selection to develop FW resistant varieties of R. sativus.

QTL Analysis of Stem Elongation and Flowering Time in Lettuce Using Genotyping-by-Sequencing.

Lettuce plants tend to undergo floral initiation by elongation of flower stalks (bolting) under high-temperature and long-day conditions, which is a serious problem for summer lettuce production. Our objective was to generate a high-density genetic map using SNPs obtained from genotyping-by-sequencing (GBS) analysis of F5 recombinant inbred lines (RILs) and to map QTLs involved in stem growth and flowering time in lettuce. A set of 127 intra-specific RIL mapping populations derived from a cross between two varieties, green and red leaf lettuce, were used to identify QTLs related to the number of days from sowing to bolting (DTB), to flowering of the first flower (DTF), to seed-setting of the first flower (DTS), and the total number of leaves (LN), plant height (PH), and total number of branches of main inflorescence (BN) for two consecutive years. Of the 15 QTLs detected, one that controls DTB, DTF, DTS, LN, and PH detected on LG 7, and another QTL that controls DTF, DTS, and PH detected on LG 1. Analysis of the genomic sequence corresponding to the QTL detected on LG 7 led to the identification of 22 putative candidate genes. A consistent QTL related to bolting and flowering time, and corresponding candidate genes has been reported. This study will be valuable in revealing the genetic basis of stem growth and flowering time in lettuce.

Development of Molecular Markers for Predicting Radish (Raphanus sativus) Flesh Color Based on Polymorphisms in the RsTT8 Gene.

Red radish (Raphanus sativus L.) cultivars are a rich source of health-promoting anthocyanins and are considered a potential source of natural colorants used in the cosmetic industry. However, the development of red radish cultivars via conventional breeding is very difficult, given the unusual inheritance of the anthocyanin accumulation trait in radishes. Therefore, molecular markers linked with radish color are needed to facilitate radish breeding. Here, we characterized the RsTT8 gene isolated from four radish genotypes with different skin and flesh colors. Sequence analysis of RsTT8 revealed a large number of polymorphisms, including insertion/deletions (InDels), single nucleotide polymorphisms (SNPs), and simple sequence repeats (SSRs), between the red-fleshed and white-fleshed radish cultivars. To develop molecular markers on the basis of these polymorphisms for discriminating between radish genotypes with different colored flesh tissues, we designed four primer sets specific to the RsTT8 promoter, InDel, SSR, and WD40/acidic domain (WD/AD), and tested these primers on a diverse collection of radish lines. Except for the SSR-specific primer set, all primer sets successfully discriminated between red-fleshed and white-fleshed radish lines. Thus, we developed three molecular markers that can be efficiently used for breeding red-fleshed radish cultivars.

Establishment of a Rapid Micropropagation System for Kaempferia parviflora Wall. Ex Baker: Phytochemical Analysis of Leaf Extracts and Evaluation of Biological Activities.

This study aimed to establish a rapid in vitro plant regeneration method from rhizome buds of Kaempferia parviflora to obtain the valuable secondary metabolites with antioxidant and enzyme inhibition properties. The disinfection effect of silver oxide nanoparticles (AgO NPs) on rhizome and effects of plant growth regulators on shoot multiplication and subsequent rooting were investigated. Surface sterilization of rhizome buds with sodium hypochlorite was insufficient to control contamination. However, immersing rhizome buds in 100 mg L-1 AgO NPs for 60 min eliminated contamination without affecting the survival of explants. The number of shoots (12.2) produced per rhizome bud was higher in Murashige and Skoog (MS) medium containing 8 µM of 6-Benzyladenine (6-BA) and 0.5 µM of Thidiazuron (TDZ) than other treatments. The highest number of roots (24), with a mean root length of 7.8 cm and the maximum shoot length (9.8 cm), were obtained on medium MS with 2 µM of Indole-3-butyric acid (IBA). A survival rate of 98% was attained when plantlets of K. parviflora were acclimatized in a growth room. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to determine the chemical profile of K. parviflora leaf extracts. Results showed that several biologically active flavonoids reported in rhizomes were also present in leaf tissues of both in vitro cultured and ex vitro (greenhouse-grown) plantlets of K. parviflora. We found 40 and 36 compounds in in vitro cultured and ex vitro grown leaf samples, respectively. Greenhouse leaves exhibited more potent antioxidant activities than leaves from in vitro cultures. A higher acetylcholinesterase inhibitory ability was obtained for greenhouse leaves (1.07 mg/mL). However, leaves from in vitro cultures exhibited stronger butyrylcholinesterase inhibitory abilities. These results suggest that leaves of K. parviflora, as major byproducts of black ginger cultivation, could be used as valuable alternative sources for extracting bioactive compounds.

Micropropagation and Subsequent Enrichment of Carotenoids, Fatty Acids, and Tocopherol Contents in Sedum dasyphyllum L.

A promising micropropagation protocol has been systematically established and demonstrated for the enhanced production of carotenoids, tocopherol and fatty acids in shoot tissues of Sedum dasyphyllum. Shoot tip explants were grown on Murashige and Skoog (MS) medium. Different concentrations of N6-benzyladenine (BA) or thidiazuron (TDZ) alone or in combination with α-naphthaleneacetic acid (NAA) were tested in order to stimulate multiple shoot production. Ideal shoot induction (100%) and maximized shoot numbers (36.4) were obtained on explants cultured on media incorporated with 2 µM BA and 1 µM NAA combinations. The in vitro-developed shoots rooted best on half-strength MS media incorporated with 2 µM indole 3-butyric acid. Plantlets were effectively acclimatized in the greenhouse with 100% survival rate. The composition and contents of bioactive compounds such as carotenoids, tocopherol and fatty acids in shoot tissues of S. dasyphyllum were investigated using HPLC and GC-MS. The most abundant carotenoid in the shoot tissue was all-E-lutein (40.3-70.5 µg g-1 FW) followed by 9'-Z-neoxanthin (5.3-9.9 µg g-1 FW), all-E-violaxanthin (4.4-8.2 µg g-1 FW), and all-E-ß-carotene (1.6-3.6 µg g-1 FW). The α-tocopherol contents of in vitro-raised shoots was 6.5-fold higher than shoots of greenhouse-grown plants. The primary fatty acids found in shoot tissues were α-linolenic acid (32.0-39.3%), linoleic acid (27.4-38.2%), palmitic acid (13.3-15.5%), and stearic acid (5.2-12.2%). In all, summarizing the findings, the micropropagated S. dasyphyllum showed significant enrichment of valuable bioactive carotenoids (92.3 µg g-1 FW), tocopherols (14.6 µg g-1 FW), and α-linolenic acid (39.3%) compared to their greenhouse counterparts. The protocol demonstrated here could be applied for the mass propagation and production of enhanced bioactive compounds from S. dasyphyllum with credibility.

The complete chloroplast genome sequence of Oryza sativa aus-type variety Nagina-22 (Poaceae).

Rice (Oryza sativa) is the predominant staple food crop belonging to the Poaceae family. In this study, complete chloroplast genome sequence of O. sativa aus-type variety Nagina-22 was characterized through de novo assembly. The genome is a circular DNA molecule of 134,503 bp and has typical quadripartite structures including large single copy region (80,548 bp), small single copy region (12,347 bp), and a pair of inverted repeats (20,804 bp). A total of 120 genes were predicted in the genome, including 77 protein-coding genes, 8 open reading frame genes, 31 tRNA genes, and 4 rRNA genes. Phylogenetic analysis confirmed a close taxonomical relationship with O. sativa ssp. Indica species.