Ultrastructural and molecular implications of ecofriendly made silver nanoparticles treatments in pea (Pisum sativum L.)

Background Silver nanoparticles (AgNPs) are the most widely used nanomaterial in agricultural and environmental applications. In this study, the impact of AgNPs solutions at 20 mg/L, 40 mg/L, 80 mg/L, and 160 mg/L on cell ultrastructure have been examined in pea (Pisum sativum L) using a transmission electron microscope (TEM). The effect of AgNPs treatments on the α, β esterase (EST), and peroxidase (POX) enzymes expression as well as gain or loss of inter-simple sequence repeats (ISSRs) markers has been described. Results Different structural malformations in the cell wall and mitochondria, as well as plasmolysis and vacuolation were recorded in root cells. Damaged chloroplast and mitochondria were frequently observed in leaves and the osmiophilic plastoglobuli were more observed as AgNPs concentration increased. Starch grains increased by the treatment with 20 mg/L AgNPs. The expressions of α, β EST, and POX were slightly changed but considerable polymorphism in ISSR profiles, using 17 different primers, were scored indicating gain or loss of gene loci as a result of AgNPs treatments. This indicates considerable variations in genomic DNA and point mutations that may be induced by AgNPs as a genotoxic nanomaterial. Conclusion AgNPs may be used to induce genetic variation at low concentrations. However, considerations should be given to the uncontrolled use of nanoparticles and calls for evaluating their impact on plant growth and potential genotoxicity are justified.

Analysis of Hypericum accessions by DNA fingerprinting and flow cytometry

Hypericum perforatum, H. umbellatum, H. maculatum, and H. hircinum accessions originating from botanical gardens across Europe were examined by flow cytometry and molecular markers. 2C DNA content of 17 Hypericum perforatum accessions (Hp) and the H. perforatum cultivar Topaz amounted to between 1.56 pg and 1.62 pg. In four Hp accessions some individual plants were found with a DNA content corresponding to 6Cx (2.34 - 2.39 pg). All plants of accession Hp8 showed a DNA content of 6Cx (2.41 pg). In root tips of Hp plants with an average DNA amount of 1.58 pg, 32 chromosomes were detected, corresponding to 2n = 4x. This is the first ploidy and/or DNA content report for H. umbellatum, H. maculatum and H. hircinum. H. umbellatum and H. maculatum, each contained 0.76 pg DNA and 16 chromosomes were counted. The 2C DNA content of H. hircinum was 1.00 pg with the best metaphase plate revealing 32 chromosomes. Additionally, a combined marker analysis, based on inter-simple sequence repeats (ISSR) and sequence related amplified polymorphism (SRAP), was conducted to gain a better understanding of diversity especially within the accessions of H. perforatum. A total of 27 (11 ISSR and 16 SRAP) primer combinations were screened, showing 699 bands, of which 661 were polymorphic. UPGMA clustering revealed that accessions from the same geographic area tended to be more closely related, while H. maculatum was grouped separately from all H. perforatum accessions. Both methods have shown similar sensitivities in detecting the genetic diversity of the analyzed genotypes. Our results may be useful for Hypericum breeding programs and the development of effective conservation strategies.

Molecular identification and genetic relationships among Alcea (Malvaceae) species by ISSR Markers: A high value medicinal plant

Alcea L. is one of the largest genera of Malvaceae family with nearly 70 species worldwide mainly distributed in SW Asia. According to the latest revision of the family, it is represented by 34 species in the Flora of Iran, among them, 15 species are endemic. It is tough to accurate germplasm/ plant recognition by using morphological characteristics because of its propagation, growing and using. We conducted a molecular data analysis on these plant species due to their importance. We examined 156 plants from 14 species in 16 regions that were selected randomly for this investigation. It has been 119 polymorphic bands (94.33%) were resulted from 128 bands of 10 primers in amplification of genomic DNA. ISSR primers have a great capacity to detect polymorphic loci among Alcea species, as evidenced by the high average PIC and MI values found. The genetic similarity of 14 species was calculated and ranged between 0.635 to 0.990. Inter-Simple sequence repeats (ISSR) markers research revealed that Alcea tarica Pakravan & Ghahreman and Alcea kopetdaghensis lljin had the least similarity, while Alcea semnanica Pakravan and Alcea mazandaranica Pakravan & Ghahreman had the most. The current study attempts to answer three questions: 1) can ISSR markers identify Alcea species? 2) what is the genetic structure of these taxa in Iran? and 3) what is the inter-relationship between these taxa? The current study discovered that ISSR markers can be used to identify species.

Picloram-induced enhanced callus-mediated regeneration, acclimatization, and genetic clonality assessment of gerbera

Background Gerbera jamesonii Bolus ex Hooker f. (African daisy) is listed among the top five most important ornamental plants in the global floricultural industry. To satisfy its demand, the floriculture industry relies on reproducible and effective propagation protocol while retaining the genetic uniformity of G. jamesonii. The present study, for the first time, reports the potential of picloram for enhanced induction of organogenic calli from leaves of G. jamesonii and its high-frequency indirect regeneration. Results The fastest induction of calli with maximum fresh and dry weight was recorded in the Murashige and Skoog (MS) semisolid medium supplemented with 1 mg/l picloram. In addition, callus induction was observed in 2,4-dichlorophenoxy acetic acid- and α-napthaleneaceticacid-supplemented media but with delayed response and reduced fresh and dry weight. The proliferated calli were transferred to shoot induction media containing MS salt and 0.5–1 mg/l N6-benzylaminopurine, kinetin, or thidiazuron. A mean number of ~6 shoots per callus were developed after 5 days of culture in the MS medium supplemented with 1 mg/l kinetin, with a mean length of 5.2 cm. Successful rooting of shoots was achieved in the MS medium fortified with 1.5 mg/l indole-3-acetic acid, wherein the earliest root initiation (~5 days), as well as the maximum number (~9) and length (~4.8 cm) of roots, were recorded. Complete plantlets were primarily acclimatized in sand before being transferred to a mixed substrate (of soil, sand, tea leaf waste, and cow urine) that secured >90% survival and further growth of the plantlets. Eventually, clonal fidelity of the in vitro regenerants assessed via inter-simple sequence repeats (ISSR) primers exhibited a monomorphic banding patterns that suggested genetic integrity within the plantlets as well as with their mother plant. Conclusions The results of the present study should be of interest for commercial propagation and mutagenesis- as well as genetic transformation-related research.

GENETIC DIVERSITY OF LIMA BEAN (PHASEOLUS LUNATUS L.) FROM TIMOR ISLAND BASED ON MOLECULAR MARKER INTER-SIMPLE SEQUENCE REPEATS (ISSR)

Lima bean (Phaseolus lunatus L.) is one of the important legume vegetables in Indonesia. However, genetic information for these plants is still minimalized, especially on Timor Island. This study aims to analyze the genetic diversity of lima beans from Timor Island based on ISSR molecular markers. A total of 4 accessions of lima beans were analyzed using 3 ISSR primers to produce 15 polymorphic bands with an average of 68.18% polymorphism. The cluster analysis results use the Unweighted Pair Group Methods using Arithmetic averages (UPGMA) method to create a dendrogram that produces two main clusters. There were plain seed and pattern seed group with a similarity coefficient of 0.52. These results indicated that the genetic variation of the lima beans from Timor Island was high. Moreover, the result provides a sutable method for evaluating the genetic diversity of lima beans using the ISSR marker and important information of future lima bean breeding programs.

Genetic Diversity of Toxigenic Fusarium Verticillioidesassociated with Maize Grains in India

Genetic diversity of Fusarium species associated with maize grains were analyzed from ten states of India. Fusarium verticillioides (88.52%), F. coffeatum (6.55%), F. foetens (3.27%) and F. euwallaceae (1.63%) were recovered from the maize samples. A phylogenetic analysis was performed based on the translation elongation factor 1 alpha (TEF1-α) gene sequence formed five evolutionary lineages (EL) that showed both inter- and intra-species variability. A In-vitro pathogenicity study confirms that among the 61 strains, 51% exhibited high to moderate virulence to the maize, and produced one or more mycotoxins. The genetic diversity of F. verticillioides were evaluated using Inter simple sequence repeats (ISSR) fingerprints. The ISSR fingerprints (AG)8C and (AG)8G shown 252 and 368 microsatellites sites, respectively, in the genome of F. verticillioides. The (AG)8C fingerprints differentiated the pathogenic and non-pathogenic clustering of F. verticillioides. However, Simpson’s (SID), and Shannon’s (H) index showed a wide range of diversity within the F. verticillioides. The pleomorphic nature of these strains role in host pathogen interactions and adoptive potential to develop disease to crop plants. Toxigenic potential of F. verticillioides was evaluated for the presence of FUM1and FUM13 genes involved in fumonisins B1 (FB1) biosynthesis, and further confirmed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) showed 91% strains were FB1 producers. The current finding demonstrated that most F. vertcillioides strains showed a wide range of genetic diversity with varied toxigenic and pathogenic potentials. Thus, there is an urgent need to develop an effective resistant breeding program in maize, and further monitoring is necessary for the post-harvest losses of grains.

Assessment of Genetic Diversity of Rheum species (Endangered Medicinal Herb of Indian Himalayan Region) using Molecular Markers

The Rheum species are important medicinal plants that are facing extinction due to their unplanned development and overexploitation by pharmaceutical industries. DNA polymorphisms are not prone to environmental modifications, thus they are widely used for the identification and characterization of plants. The use of different molecular markers has enabled the researchers for the valuation of genetic variability and diversity in its natural zone of distribution. The conventional approach may take several years to yield this information. For the estimation of molecular and genetic variations in geographical zone of distribution, various molecular markers technique are available like RAPD (Randomly Amplified Polymorphic DNA), RFLP (Restriction fragment length polymorphism), ISSR (Inter-Simple Sequence Repeats), SSR and AFLP. The uses of different molecular markers for the study of genetic diversity have been discussed in the review.

Study of Genetic Diversity Among Crataegus Species (Hawthorn) Using ISSR Markers in Northwestern of Iran

Background: Crataegus spp. (Hawthorn) are used for horticultural and medicinal purposes. Objectives: In the present study, the genetic relationships among the five Iranian Crataegus species were evaluated by Inter Simple Sequence Repeats (ISSR) molecular marker Methods: PCR reactions were performed using six primers (811, 827, 834, 845, 856, and 868) and genetic similarity was calculated based on Jacquard’s similarity coefficient. Results: Six primers generated 79 products in total, of which 71 were polymorphic (89.9%), with an average of 13.1 bands per primer. The percentage of polymorphic bands ranged from 77 to 100. Primer 856 produced the highest number of bands, while the lowest was generated by primer 845. The Jacquard’s similarity coefficient, derived from ISSR marker analysis, ranged from 0.164 to 0.337, indicating high genetic variation among Crataegus species in Iran. Conclusion: This study provides important data for identifying species relationships and helps develop plant breeding strategies to improve the medicinal properties of this genus in the future.