Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant.

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an ever-increasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg/L BAP, 0.3 mg/L GA3, and 0.3 mg/L ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity. https://doi.org/10.54680/fr24210110412.

Involvement of Dynamic Adjustment of ABA, Proline and Sugar Levels in Rhizomes in Effective Acclimation of Solidago gigantea to Contrasting Weather and Soil Conditions in the Country of Invasion.

Giant goldenrod (Solidago gigantea Aiton) is one of the most invasive plant species occurring in Europe. Since little is known about the molecular mechanisms contributing to its invasiveness, we examined the natural dynamics of the content of rhizome compounds, which can be crucial for plant resistance and adaptation to environmental stress. We focused on rhizomes because they are the main vector of giant goldenrod dispersion in invaded lands. Water-soluble sugars, proline, and abscisic acid (ABA) were quantified in rhizomes, as well as ABA in the rhizosphere from three different but geographically close natural locations in Poland (50°04'11.3″ N, 19°50'40.2″ E) under extreme light, thermal, and soil conditions, in early spring, late summer, and late autumn. The genetic diversity of plants between locations was checked using the random amplified polymorphic DNA (RAPD) markers. Sugar and proline content was assayed spectrophotometrically, and abscisic acid (ABA) with the ELISA immunomethod. It can be assumed that the accumulation of sugars in giant goldenrod rhizomes facilitated the process of plant adaptation to adverse environmental conditions (high temperature and/or water scarcity) caused by extreme weather in summer and autumn. The same was true for high levels of proline and ABA in summer. On the other hand, the lowering of proline and ABA in autumn did not confirm the previous assumptions about their synthesis in rhizomes during the acquisition of frost resistance by giant goldenrod. However, in the location with intensive sunlight and most extreme soil conditions, a constant amount of ABA in rhizomes was noticed as well as its exudation into the rhizosphere. This research indicates that soluble sugars, proline, and ABA alterations in rhizomes can participate in the mechanism of acclimation of S. gigantea to specific soil and meteorological conditions in the country of invasion irrespective of plant genetic variation.

The potential of gamma irradiation on antioxidant capacity and genomic alterations in Calendula officinalis.

There are lines of evidence that ionizing radiations such as gamma rays can cause different biological effects on plants. Marigold (Calendula officinalis L.) is a member of the family Asteraceae. It possesses profound amounts of active ingredients. The aim of this study was to evaluate the changes imposed upon different dose levels of gamma radiation on some features of Calendula officinalis such as antioxidant activity, total phenolic compounds and flavonoid contents, antibacterial activity and genomic alterations. Calendula officinalis seeds were exposed to different doses of Gamma radiation (0, 10, 15, 20 and 25 GY). Total phenolics, flavonoids, antioxidant activity (measured by DPPH assay) using methanolic extracts of plants and antibacterial activity measured by the disc diffusion assay showed significant differences to the control samples. The samples treated with 10 GY gamma rays showed the highest total phenol and flavonoid contents. Antioxidant activity significantly differed between Gamma rays dose levels and it was the highest at 25 GY. Four bacterial strains including E. coli, Bacillus subtilis and Pseudomonas aeroginosa were used for the antibacterial assay. Extracts from plants treated with 25 GY gamma rays showed the highest antibacterial activity against the 4 bacterial strains. Random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers were used to study the genetic variation. The polymorphism information content (PIC) for RAPD primers ranged from 3% to 13% and ranged from 6 to 13% for ISSR primers. Results indicated that ISSR markers were more efficient than RAPD markers, as they detected 25.57% polymorphic DNA bands compared to 21.31% polymorphism for RAPD markers.

Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products.

BACKGROUND: Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name "Brahmi'' being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. METHODS AND RESULTS: This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls (in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. CONCLUSION: SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica. However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.

Quantum leap in the light of molecular elucidation of garlic genome.

Garlic, a popular vegetable cum condiment is known widely for its health benefits, pharmacological properties and in curing several pathological conditions. This compelling horticultural bulb crop is propagated asexually from individual bulbils or cloves. It is an obligate apomict that lost its fertility and blooming potential long ago and probable reason for evolution from fertility to sterility to greater contiguity of human selection to asexual propagules as they are used in culinary as and when required. The crop is likely to be sterile owing to nutritional competition between topsets, pollen degeneration, chromosomal deletion, irregular chromosomal pairing and abnormal meiosis during gametogenesis and thus curbing genetic variation is needed utmost for its improvement. With asexual reproduction, molecular studies are challenging due to its expected and complex genome. Alongside classical molecular markers like RAPDs, AFLPs, SRAPs, SSRs, and isozymes; recent high-throughput genotyping-by-sequencing (GBS) approaches like DArTseq has allowed characterization, mapping, whole-genome profiling, DNA fingerprinting among others in garlic. However, in recent years, biotechnological tools, genetic transformation via biolistic or Agrobacterium tumefaciens, polyploidization or chromosomal doubling have emerged as a potent breeding tool in enabling the improvement of vegetatively propagated plants such as garlic. In recent times biological responses of garlic and its compounds have been studied using epigenomics, proteomics and transcriptomics by researchers in preclinical studies instigating the biological effects of garlic and such gene expression revealed many early mechanistic events which may clinically underlie important health benefits pertaining to garlic intake. This review thus encompasses efforts achieved till present date towards elucidation of garlic genome with regard to molecular, biotechnological analysis and gene expression in terms of in vitro and in vivo studies.

In vitro genotoxicity assessment of biosynthesized zinc oxide nanoparticles.

There are various studies on the toxicological potentials of conventionally synthesized zinc oxide (ZnO) nanoparticles, which are useful tools for many medical applications. However, knowledge about the biologically synthesized ones is still limited. In this study, the potential of producing ZnO nanoparticles via a green synthesis method, which enables safer, environmentally, economical and controlled production by using the Symphoricarpos albus L. plant, was investigated. For this purpose, aqueous extract was obtained from the fruits of the plant and reacted with zinc nitrate precursor. Characterization of the synthesized product was carried out by SEM and EDAX analyzes. In addition, the biosafety of the product was also investigated by using the Ames/Salmonella, E. coli WP2, Yeast DEL, seed germination, and RAPD test systems. The results obtained from SEM studies showed that spherical nanoparticles with an average diameter of 30 nm were synthesized as a result of the reaction. EDAX findings confirmed that these nanoparticles were composed of Zn and O elements. On the other hand, according to the findings of the biocompatibility tests, the synthesized nanoparticle did not show any toxic and genotoxic effects up to a concentration of 640 µg/ml in any of the test systems. Accordingly, considering the findings of our study, it was concluded that the aqueous extract of S. albus fruits can be used for the green synthesis of ZnO nanoparticles, the products obtained successfully passed the biocompatibility tests in our study, and additionally, more comprehensive biocompatibility tests should be performed before industrial scale production.

Biosynthesis of Silver Nanoparticles and Exploring Their Potential of Reducing the Contamination of the In Vitro Culture Media and Inducing the Callus Growth of Rumex nervosus Explants.

Among biological methods, green synthesis of the nanomaterials using plant extracts was shown to be an environmentally friendly, economical, and simple approach. In the current study, the biogenic synthesis of silver nanoparticles (AgNPs) was achieved using the leaf extract of Hibiscus tiliaceus, in order to prevent the contamination of the tissue culture media and induce callus growth. The nanostructures of the fabricated AgNPs were characterized using UV-visible spectroscopy, Fourier transform infra-red spectra (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), zeta size, and zeta potential techniques. Our results indicate that The UV-vis spectrum of AgNPs exhibited an absorption band at 415 nm. The FTIR analysis identified the functional groups which could involve in the reduction of silver ions to AgNPs, this was also confirmed by the (hkl) diffraction peaks in the XRD diffractogram. Moreover, the TEM analysis showed a spherical nanoparticle with a size ranging from 21 and 26 nm. Thereafter, the potential antibacterial and antifungal activity of the biogenic AgNPs was evaluated against Bacillus pumilus and Alternaria alternata which were isolated from the in vitro culture media and identified based on 16S rDNA and ITS rDNA sequences, respectively. The results showed that the AgNPs significantly inhibited the growth of Alternaria alternata and Bacillus pumilus at all applied concentrations (5, 10, 20 and 40 mg/L). Compared to the control more fungal radial growth reduction (42.59%,) and bacterial inhibition (98.12%) were registered in the plates containing high doses of AgNPs (40 mg/L). Using Rumex nervosus explants, the biosynthesized AgNPs were tested for their impact to promote callus growth. The obtained results showed a significant effect of AgNPs on callus fresh weight at all applied doses. Moreover, AgNPs treatments showed a polymorphism of 12.5% which was detected by RAPD markers. In summary, the results revealed that AgNPs (40 mg/L) can be effectively added to the in vitro culture media for reducing microbial contamination and improving callus growth while greatly maintaining its genetic stability.

Bacteriocin production and technological properties of Enterococcus mundtii and Enterococcus faecium strains isolated from sheep and goat colostrum.

Enterococci are lactic acid bacteria (LAB) that play a role in the aroma formation, maturation, and sensory development of fermented foods such as meat and dairy products. They also contribute to the improvement of the extended shelf life of fermented foods by producing bacteriocin. The aim of this study was to isolate bacteriocin-producing LAB from sheep and goat colostrum, to characterize the bacteriocin-producing strains, and determine the technological properties of the strains. A total of 13 bacteriocin-producing LAB was isolated and identified as 11 Enterococcus mundtii and two Enterococcus faecium. The strains were found to be genetically different from each other by phylogenetic analysis of 16S rRNA gene sequences and random amplified polymorphic-DNA (RAPD-PCR). It has been determined that bacteriocins show activity in a wide pH range and are resistant to heat, lose their activity with proteolytic enzymes and α-amylase, but are resistant to detergents. While the presence of the munKS gene was detected in all of the strains, it was determined that E. faecium HC121.4, HC161.1, E. mundtii HC147.1, HC166.5, and HC166.8 strains contained multiple enterocin genes. Trisin-SDS-PAGE analysis revealed two active protein bands of approximately 5.1 and 5.5 kDa in E. faecium HC121.4 and one active protein band with a weight of approximately 4.96 kDa in other strains. E. mundtii strains and E. faecium HC161.1 were identified as mundticin KS producers, and E. faecium HC121.4 was defined as an enterocin A and B producer. Except for E. mundtii HC166.8, acid production of strains was found to be slow at 6 h and moderate at 24 h. None of them showed extracellular proteolytic and lipolytic activities. It was found that the strains had esterase, esterase lipase, leucine arylamidase, acid phosphatase, and naphthol-AS-Bl-phosphohydrolase activities, while protease activities were low and peptidase activities were high. In conclusion, bacteriocin producer 13 Enterococcus strains isolated from sheep and goat colostrum were found to have the potential to be included in starter culture combinations.

In vitro propagation of Codonopsis pilosula (Franch.) Nannf. using apical shoot segments and phytochemical assessments of the maternal and regenerated plants.

BACKGROUND: Codonopsis pilosula (Franch.) Nannf. is a medicinal plant traditionally used in China, Korea, and Japan to treat many diseases including poor gastrointestinal function, low immunity, gastric ulcers, and chronic gastritis. The increasing therapeutic and preventive use of C. pilosula has subsequently led to depletion of the natural populations of this species thus necessitating propagation of this important medicinal plant. Here, we developed an efficient and effective in vitro propagation protocol for C. pilosula using apical shoot segments. We tested various plant tissue culture media for the growth of C. pilosula and evaluated the effects of plant growth regulators on the shoot proliferation and rooting of regenerated C. pilosula plants. Furthermore, the tissues (roots and shoots) of maternal and in vitro-regenerated C. pilosula plants were subjected to Fourier-transform near-infrared (FT-NIR) spectrometry, Gas chromatography-mass spectrometry (GC-MS), and their total flavonoids, phenolics, and antioxidant capacity were determined and compared. RESULTS: Full-strength Murashige and Skoog (MS) medium augmented with vitamins and benzylaminopurine (1.5 mg·L-1) regenerated the highest shoot number (12 ± 0.46) per explant. MS medium augmented with indole-3-acetic acid (1.0 mg·L-1) produced the highest root number (9 ± 0.89) and maximum root length (20.88 ± 1.48 mm) from regenerated C. pilosula shoots. The survival rate of in vitro-regenerated C. pilosula plants was 94.00% after acclimatization. The maternal and in vitro-regenerated C. pilosula plant tissues showed similar FT-NIR spectra, total phenolics, total flavonoids, phytochemical composition, and antioxidant activity. Randomly amplified polymorphic DNA (RAPD) test confirmed the genetic fidelity of regenerated C. pilosula plants. CONCLUSIONS: The proposed in vitro propagation protocol may be useful for the rapid mass multiplication and production of high quality C. pilosula as well as for germplasm preservation to ensure sustainable supply amidst the ever-increasing demand.

Assessment of health risk, genotoxicity, and thiol compounds in Trigonella foenum-graecum (Fenugreek) under arsenic stress.

Arsenic (As) traces have been reported worldwide in vegetables and crops cultivated in As-polluted soils. Being carcinogenic, the presence of As in edibles is of great concern as it ultimately reaches humans and animals through the food chain. Besides, As toxicity adversely affects the growth, physiology, metabolism, and productivity of crops. In the present study, Trigonella foenum-graecum (Fenugreek) was exposed to the As stress (0, 50, 100, and 150 µM sodium arsenate) for a week. Further, evaluation of As accumulation in roots and shoots, magnitude and visualization of oxyradicals, and thiol-based defence offered by Fenugreek was assessed. The root and leaf accumulated 258-453 µg g-1 dry wt (DW) and 81.4-102.1 µg g-1 DW of As, respectively. An arsenic-mediated decline in the growth index and increase in oxidative stress was noted. Arsenic stress modulated the content of thiol compounds; especially cysteine content increased from 0.36 to 0.43 µmole g-1 FW protein was noted. Random Amplified Polymorphic DNA (RAPD)-based analysis showed DNA damage in As-treated plants. Health risk assessment parameters showed that As concentration in the consumable plant shoot was below the critical hazard level (hazard quotient < 1). Moreover, T. foenum-graecum showed varied responses to As-induced oxidative stress with applied concentrations (150 µM being more toxic than lower concentrations). In addition, the RAPD profile and level of thiol compounds were proved significant biomarkers to assess the As toxicity in plants. The conclusion of this study will help users of fenugreek to have a clue and create awareness regarding the consumption.

Pulsed Light Mutagenesis of the Willow Bracket Mushroom, Phellinus igniarius (Agaricomycetes), for Enhanced Production of Flavonoids, Laccase, and Fermentation Biomass.

Phellinus igniarius is a medicinal fungus possessing potent therapeutic activity due to the polysaccharides, polyphenols, flavonoids, and other secondary metabolites they contain. Laccases are crucial enzymes involved in lignin degradation in Ph. igniarius and offer great potential to accomplish several bioprocesses. To generate Ph. igniarius strains with high biomass, flavonoid, and laccase activity, we used pulsed light (PL) technology for mutagenesis of Ph. igniarius protoplasts and screened for mutants with high biomass, flavonoid, and laccase activity. At the irradiation power of 100 J, treated distance 8.5 cm, irradiation frequency was 0.5 s/time, three times treatments, after five generations of selection, three mutants were obtained with higher biomass production. Compared with control, the mycelium biomass and the flavonoid production of the screened mutant strain QB72 were increased 20.87% and 53.51%, respectively. The total amount of the accumulated extracellular laccase of the QB72 in the first 6 and 8 days increased 23.38% and 22.37% respectively, and over the total 16 days it increased 9.62%. In addition, RAPD analysis results indicated that the genetic materials of the mutant QB72 were altered. PL mutagenesis method has great potential for developing strains, especially Phellinus.

Genetic diversity and iron metabolism of Staphylococcus hominis isolates originating from bovine quarter milk, rectal feces, and teat apices.

Staphylococcus hominis, a member of the non-aureus staphylococci (NAS) group, is part of the human and animal microbiota. Although it has been isolated from multiple bovine-associated habitats, its relevance as a cause of bovine mastitis is currently not well described. To successfully colonize and proliferate in the bovine mammary gland, a bacterial species must be able to acquire iron from host iron-binding proteins. The aims of this study were (1) to assess the genetic diversity of S. hominis isolated from bovine quarter milk, rectal feces, and teat apices, and (2) to investigate the capacity of bovine S. hominis isolates belonging to these different habitats to utilize ferritin and lactoferrin as iron sources. To expand on an available collection of bovine S. hominis isolates (2 from quarter milk, 8 from rectal feces, and 19 from teat apices) from one commercial dairy herd, a subsequent single cross-sectional quarter milk sampling (n = 360) was performed on all lactating cows (n = 90) of the same herd. In total, 514 NAS isolates were recovered and identified by MALDI-TOF mass spectrometry; the 6 most prevalent NAS species were S. cohnii (33.9%), S. sciuri (16.7%), S. haemolyticus (16.3%), S. xylosus (9.6%), S. equorum (9.4%), and S. hominis (3.5%). A random amplified polymorphic DNA (RAPD) analysis was performed on 46 S. hominis isolates (19 from quarter milk, 8 from rectal feces, and 19 from teat apices). Eighteen distinct RAPD fingerprint groups were distinguished although we were unable to detect the presence of the same RAPD type in all 3 habitats. One S. hominis isolate of a distinct RAPD type unique to a specific habitat (8 from quarter milk, 3 from rectal feces, and 4 from teat apices) along with the quality control strain Staphylococcus aureus ATCC 25923 and 2 well-studied Staphylococcus chromogenes isolates ("IM" and "TA") were included in the phenotypical iron test. All isolates were grown in 4 types of media: iron-rich tryptic soy broth, iron-rich tryptic soy broth deferrated by 2,2'-bipyridyl, and deferrated tryptic soy broth supplemented with human recombinant lactoferrin or equine spleen-derived ferritin. The growth of the different strains was modified by the medium in which they were grown. Staphylococcus chromogenes TA showed significantly lower growth under iron-deprived conditions, and adding an iron supplement (lactoferrin or ferritin) resulted in no improvement in growth; in contrast, growth of S. chromogenes IM was significantly recovered with iron supplementation. Staphylococcus hominis strains from all 3 habitats were able to significantly utilize ferritin but not lactoferrin as an iron source to reverse the growth inhibition, in varying degrees, caused by the chelating agent 2,2'-bipyridyl.

Assessing the genetic fidelity of somatic embryo-derived plantlets of finger millet by random amplified polymorphic DNA analysis.

Finger millet [Eleusine coracana (L.) Gaertn.] is an important cereal because of its mineral-nutrition value. With the increasing demand, there is a pressing need to conserve it through biotechnological approaches. High-frequency somatic embryogenesis from seed-derived callus of E. coracana was developed on Murashige-Skoog (MS) medium supplemented with a combination of auxins [Indole-3-acetic acid (IAA), 2,4-Dichlorophenoxy acetic acid (2,4-D)] and cytokinins [6-Benzylaminopurine (BAP), kinetin (KN)] in different concentrations, ranging from 0.1 to 5.0 mg L-1. Seeds cultured on this medium produced three different types of primary callus. Type I callus was very compact and dark brown, type II callus was light brownish and type III callus appeared whitish and light brown. All three types of calli had differential proliferation responses. Type II compact brown calli were obtained on the MS medium supplemented with 1.0 and 1.5 mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5 mg kinetin L-1. Friable yellowish embryogenic calli with a large number of somatic embryos were developed within 60 days after being transferred to auxins and cytokinin (1.0 and 1.5 mg 2,4-Dichlorophenoxy acetic acid L-1 and 0.5 mg Kinetin L-1) along with 200 mg casein hydrolysate L-1. Germination of somatic embryos on a half-strength MS medium supplemented with 0.1% Kinetin led to the development of healthy plantlets within 30 days. Genetic fingerprinting using random amplified polymorphic DNA (RAPD) revealed high levels of genetic fidelity. The study provides methods and hormonal concentrations required to develop somatic embryos in E. coracana for its genetic improvement and conservation.

Profiling of secondary metabolites and DNA typing of three different Annona cultivars grown in Egypt.

OBJECTIVES: Natural products are often efficacious and safe alternatives to synthetic drugs. This study explored secondary leaves and bark metabolites profiles in extracts of a new Egyptian hybrid, Annona cherimola × Annona squamosa, known as Abdel Razek. This hybrid exhibited 100% similarity with A. cherimola as evidenced by random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) analyses. METHODS: Primary constituents in methanol extracts of different plant organs were identified. Extracts richest in alkaloids and polyphenolics were assessed for in vitro antioxidant activity and the most potent were further studied in vivo for treating gastric ulcer in rats. The latter activity was assessed histopathologically. RESULTS: Structural analysis with HPLC/ESI-MSn, and UPLC/HESI-MS/MS identified 63 metabolites, including seven amino acids, 20 alkaloids, 16 flavonoids, eight phenolics and other compounds. Severe stomach alteration was observed after ethanol induction in rats. Ulcer score, oxidative stress biomarkers, cell organelles biomarker enzymes, and gastrointestinal histological features improved to variable degrees after treatment with Annona Abdel Razek hybrid leaves and bark methanol extracts. CONCLUSION: Extracts of Annona Abdel Razek had showed in vitro antioxidant effect and may be promising for the treatment of gastric ulcers.

Effect of Silver Nanoparticles on the In Vitro Regeneration, Biochemical, Genetic, and Phenotype Variation in Adventitious Shoots Produced from Leaf Explants in Chrysanthemum.

Novel and unique properties of nanomaterials, which are not apparent in larger-size forms of the same material, encourage the undertaking of studies exploring the multifaced effects of nanomaterials on plants. The results of such studies are not only scientifically relevant but, additionally, can be implemented to plant production and/or breeding. This study aimed to verify the applicability of silver nanoparticles (AgNPs) as a mutagen in chrysanthemum breeding. Chrysanthemum × grandiflorum (Ramat.) Kitam. 'Lilac Wonder' and 'Richmond' leaf explants were cultured on the modified MS medium supplemented with 0.6 mg·L-1 6-benzylaminopurine (BAP) and 2 mg·L-1 indole-3-acetic acid (IAA) and treated with AgNPs (spherical; 20 nm in diameter size; 0, 50, and 100 mg·L-1). AgNPs strongly suppressed the capability of leaf explants to form adventitious shoots and the efficiency of shoot regeneration. The content of primary and secondary metabolites (chlorophyll a, chlorophyll b, total chlorophylls, carotenoids, anthocyanins, phenolic compounds) and the activity of enzymatic antioxidants (superoxide dismutase and guaiacol peroxide) in leaf explants varied depending on the AgNPs treatment and age of culture. Phenotype variations of ex vitro cultivated chrysanthemums, covering the color and pigment content in the inflorescence, were detected in one 50 mg·L-1 AgNPs-derived and five 100 mg·L-1 AgNPs-derived 'Lilac Wonder' plants and were manifested as the color change from pink to burgundy-gold. However, no changes in inflorescence color/shape were found among AgNPs-treated 'Richmond' chrysanthemums. On the other hand, the stem height, number of leaves, and chlorophyll content in leaves varied depending on the AgNPs treatment and the cultivar analyzed. A significant effect of AgNPs on the genetic variation occurrence was found. A nearly two-fold higher share of polymorphic products, in both cultivars studied, was generated by RAPD markers than by SCoTs. To conclude, protocols using leaf explant treatment with AgNPs can be used as a novel breeding technique in chrysanthemum. However, the individual cultivars may differ in biochemical response, the efficiency of in vitro regeneration, genetic variation, and frequency of induced mutations in flowering plants.

Identification of SCAR markers for genetic authentication of Dendrobium nobile Lindl.

Dendrobium nobile Lindl. is an orcid plant with important medicinal values. This is a colourful houseplant, and also a popular herb in traditional Chinese medicine (TCM). The variants of this plant from different geographic regions might be high, and in this study, we aimed to develop specific sequence characterized amplified region (SCAR) markers for the identification of specific variant of this plant. Different cultivars of D. nobile were collected from nine different places of China, and one cultivar from Myanmar. DNA materials were extracted from the plant samples, random amplified polymorphic DNA (RAPD) were developed, cloned and sequenced for the development of SCAR markers. We have developed four SCAR markers, which are specific to the cultivar from Luzhou China, and clearly distinguishable (genetically) from other cultivars. These SCAR markers are deposited in GenBank (accession number MZ417502, MZ484089, MZ417504 and MZ417505). Four SCAR markers for D. nobile are effective molecular technique to genetically identify the different cultivars or species, and this method is applicable for genetic characterization and identification of other plant species too.

Genetic Analysis of Prunus salicina L. by Random Amplified Polymorphic DNA (RAPD) and Intersimple Sequence Repeat (ISSR).

Background: Prunus salicina L. is an important fruit tree species of great economic value which is mainly distributed in the northern hemisphere. Methods: 25 samples of Prunus salicina L. were collected from 8 provinces in China, Japan, USA, and New Zealand. The genetic variations of these samples were characterized by the random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR) technique, respectively, and in combination. Results: Totally, 257 RAPD bands ranging 200∼2300 bp was found, and 81.59% of these bands were polymorphic. ISSR analysis identified 179 bands ranging 300∼2500 bp, and 87.74% of the bands were polymorphic. ISSR results showed that the similarity coefficient index between samples P10 (Maihuangli in Anhui, Chin) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest, while that between samples P10 (Maihuangli in Anhui, Chin) and P15 (Baili in Japan) was highest. Combined analysis of RAPD and ISSR demonstrated that the similarity coefficient index between samples P4 (Qiepili in Ningbo, Zhejiang, China) and P13 (Longyuanqiuli in Heilongjiang, China) was lowest, while that between samples P19 (Laroda in USA) and P20 (Red heart in USA) was highest. Conclusion: RAPD combined with ISSR analysis can be used for genetic characterization of Prunus L. species.

Using Transcriptome Analysis to Explore Gray Mold Resistance-Related Genes in Onion (Alliumcepa L.).

Gray mold disease caused by Botrytis in onions (Allium cepa L.) during growth and storage negatively affects their yield and quality. Exploring the genes related to gray mold resistance in onion and their application to the breeding of resistant onion lines will support effective and ecological control methods of the disease. Here, the genetic relationship of 54 onion lines based on random amplified polymorphic DNA (RAPD) and in vitro-cultured onion lines infected with gray mold were used for screening resistance and susceptibility traits. Two genetically related onion lines were selected, one with a resistant and one with a susceptible phenotype. In vitro gray mold infection was repeated with these two lines, and leaf samples were collected for gene expression studies in time series. Transcript sequences obtained by RNA sequencing were subjected to DEG analysis, variant analysis, and KEGG mapping. Among the KEGG pathways, 'α-linoleic acid metabolism' was selected because the comparison of the time series expression pattern of Jasmonate resistant 1 (JAR1), Coronatine-insensitive protein 1 (COI 1), and transcription factor MYC2 (MYC2) genes between the resistant and susceptible lines revealed its significant relationship with gray-mold-resistant phenotypes. Expression pattern and SNP of the selected genes were verified by quantitative real-time PCR and high-resolution melting (HRM) analysis, respectively. The results of this study will be useful for the development of molecular marker and finally breeding of gray-mold-resistant onions.

High Genetic Diversity of 16 Indian lettuce (Lactuca indica L.) Accessions from Vietnam.

<b>Background and Objective:</b> Plant genetic resources provide the raw material for crop improvement and plant breeding program largely depends on it. Therefore, the evaluation of plant genetic resources plays a critical role in crop improvement and also in conserving valuable genetic resources for the future. In this study, the genetic diversity of 16 <i>Lactuca indica</i> L. accessions collected in Vietnam was investigated by using ISSR and RAPD markers. <b>Materials and Methods:</b> Genetic diversity of 16 <i>Lactuca sativa</i> L. genotypes collected in Vietnam were evaluated using Random Amplified Polymorphic DNA (RAPD) and Inter-Simple Sequence Repeat (ISSR) molecular markers. <b>Results:</b> In this study, 42 RAPD and ISSR primers were initially used, of which 12 and 9 primers, respectively were finally selected as they produced scorable patterns. RAPD markers produced a total of 113 loci, out of which 52 loci (45.96%) were polymorphic. The average percentage of the polymorphic band for RAPD primer is 45.96% and the genetic similarity based on simple matching coefficient ranged from 69.0-94.7%. ISSR analysis detected a total of 60 loci, out of which 22 loci (36.32%) were polymorphic and the genetic similarity ranged from 56.7-95.0%. In general, ISSR markers amplified fewer loci and showed lower variation in the percentage of polymorphism compares to the RAPD assay. <b>Conclusion:</b> These results indicate that the 16 collected Indian lettuce genotypes are genetically diverse. Because of these genetic diversities, the collected genotypes could be used for preserving or crossing programs to improve this precious medicinal plant in Vietnam.

Efficient micropropagation of Thunbergia coccinea Wall. and genetic homogeneity assessment through RAPD and ISSR markers.

Thunbergia coccinea Wall. ex D. Don being a rare, ornamental and medicinal plant of India, is needed to propagate for conserving the germplasm and analyzing its phytochemical compounds in the future. A reliable protocol for direct in vitro propagation using nodal shoot meristem of T. coccinea as explant was standardized. The highest number of shoots per explant (22.17 ± 0.54) with maximum shoot length (2.36 ± 0.28) in cm was obtained in Murashige and Skoog (MS) medium supplemented with 9.70 µM of 6-furfurylaminopurine (Kinetin) and 0.053 µM of α-naphthaleneacetic acid (NAA) combination, among all the different plant growth regulators (PGR's) and concentrations tested. The aforesaid PGR's combination was optimum for axillary shoot bud induction and multiplication in T. coccinea. The best rooting was observed on the half-strength MS medium fortified with 2.68 µM NAA with the highest number of roots per shoot (3.75 ± 0.12) and maximum length (5.22 ± 0.32) in cm. All the in vitro raised plantlets were acclimatized in sterile sand and soil mixture (1:1) with a survival rate of 70% on earthen pots under greenhouse conditions. PCR-based RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeat) molecular markers were employed to determine the genetic homogeneity amongst the plantlets. Twelve (12) RAPD and nine (9) ISSR primers developed a total of 104 and 91 scorable bands, respectively. The band profiles of micropropagated plantlets were monomorphic to the mother, donor in vivo plant, and similarity values varied from 0.9542-1.000. The dendrogram generated through UPGMA (unweighted pair group method with arithmetic mean) showed 99% similarities amongst all tested plants confirming the genetic uniformity of in vitro raised plants.