Exploring the Significance of Somaclonal Variations in Horticultural Crops.

Genetic and epigenetic variations produced via cell and tissue culture open up new sources of variability intra-species which can be used to improve crops. The use of in vitro generated somaclonal variations for selecting novel variants aids in the development of novel genotypes having desirable agronomic traits that can be released as varieties or utilized for breeding purposes. Horticultural crops give higher yield and productivity per unit area than other crops, as well as provide good economic returns which have led to an increase in their potential benefits throughout time. The last three to four decades have seen the selection and release of a number of valuable somaclonal variants, many of which possess remarkable features including disease resistance, high yield, improved nutritional quality and abiotic stress tolerance. Generating somaclonal variations has given breeders a novel alternative option for obtaining genetic diversity in horticultural crops and without advanced technologies. The variations introduced through tissue culture process, methods to determine and validate genetic changes in vitro regenerated plantlets, along with prospective application of such variations in horticultural crops' improvement are reviewed in the present work.

Nano-delivery platforms for bacterial gene transformation: suitability and challenges.

Nano-scale particles (NPs) have gained increased interest as non-viral vectors for nucleic acid delivery due to their ability to penetrate through unabraded cell membranes. The previous studies performed have evaluated the nanomaterials for their microbial transformation proficiency but have not compared the relative efficacy. The present study aims to identify the most proficient nano-delivery vehicle among the chemically synthesized/functionalized non-metal oxide, metal/metal oxide, and carbon-based (carbon nanotube (CNT), graphene oxide (GO)) nanomaterial(s) (NMs) for the transformation of two gram-negative bacteria, i.e., Escherichia coli and Agrobacterium tumefaciens. The microscopy and spectroscopy studies helped to identify the interaction, adhesion patterns, transformation efficiencies, better delivery, and expression of the target gfp gene by use of NMs. Loading of pgfp on all NMs imparted protection to DNAse I attack except ZnO NPs with maximum by chitosan, layered double hydroxide (LDH), and GO NM-plasmid DNA conjugates. The CNTs and GO significantly enhanced the extra- and intra-cellular protein content, respectively, in both bacteria. However, GO and CNT significantly decreased the cell viability in a time-dependent manner while AuNPs exhibited negligible cell toxicity. Therefore, this study identified the comparative efficiency of metal/metal oxide, non-metal oxide, and carbon nanomaterials with AuNPs as the most biosafe while LDH and chitosan NPs being the most proficient alternative tools for the genetic transformation of gram-negative bacteria by simple incubation method.

Nanobiostimulant action of trigolic formulated zinc sulfide nanoparticles (ZnS-T NPs) on rice seeds by triggering antioxidant defense network and plant growth specific transcription factors.

Under a changing climate, nanotechnological interventions for climate resilience in crops are critical to maintaining food security. Prior research has documented the affirmative response of nano zinc sulfide (nZnS) on physiological traits of fungal-infested rice seeds. Here, we propose an application of trigolic formulated zinc sulfide nanoparticles (ZnS-T NPs) on rice seeds as nanobiostimulant to improve physiological parameters by triggering antioxidative defense system, whose mechanism was investigated at transcriptional level by differential expression of genes in germinated seedlings. Nanopriming of healthy rice seeds with ZnS-T NPs (50 µg/ml), considerably intensified the seed vitality factors, including germination percentage, seedling length, dry weight and overall vigor index. Differential activation of antioxidant enzymes, viz. SOD (35.47%), APX (33.80%) and CAT (45.94%), in ZnS-T NPs treated seedlings reduced the probability of redox imbalance and promoted the vitality of rice seedlings. In gene expression profiling by reverse transcription quantitative real time PCR (qRT-PCR), the notable up-regulation of target antioxidant genes (CuZn SOD, APX and CAT) and plant growth specific genes (CKX and GRF) in ZnS-T NPs treated rice seedlings substantiates their molecular role in stimulating both antioxidant defenses and plant growth mechanisms. The improved physiological quality parameters of ZnS-T NPs treated rice seeds under pot house conditions corresponded well with in vitro findings, which validated the beneficial boosted impact of ZnS-T NPs on rice seed development. Inclusively, the study on ZnS-T NPs offers fresh perspectives into biochemical and molecular reactions of rice, potentially positioning them as nanobiostimulant capable of eliciting broad-spectrum immune and growth-enhancing responses.

In-Silico Identification, Characterization and Expression Analysis of Genes Involved in Resistant Starch Biosynthesis in Potato (Solanum tuberosum L.) Varieties.

Potato (Solanum tuberosum L.), an important horticultural crop is a member of the family Solanaceae and is mainly grown for consumption at global level. Starch, the principal component of tubers, is one of the significant elements for food and non-food-based applications. The genes associated with biosynthesis of starch have been investigated extensively over the last few decades. However, a complete regulation pathway of constituent of amylose and amylopectin are still not deeply explored. The current in-silico study of genes related to amylose and amylopectin synthesis and their genomic organization in potato is still lacking. In the current study, the nucleotide and amino acid arrangement in genome and twenty-two genes linked to starch biosynthesis pathway in potato were analysed. The genomic structure analysis was also performed to find out the structural pattern and phylogenetic relationship of genes. The genome mining and structure analysis identified ten specific motifs and phylogenetic analysis of starch biosynthesis genes divided them into three different clades on the basis of their functioning and phylogeny. Quantitative real-time PCR (qRT-PCR) of amylose biosynthesis pathway genes in three contrast genotypes revealed the down-gene expression that leads to identify potential cultivar for functional genomic approaches. These potential lines may help to achieve higher content of resistant starch.

Genome-wide identification and characterization of flowering genes in Citrus sinensis (L.) Osbeck: a comparison among C. Medica L., C. Reticulata Blanco, C. Grandis (L.) Osbeck and C. Clementina.

BACKGROUND: Flowering plays an important role in completing the reproductive cycle of plants and obtaining next generation of plants. In case of citrus, it may take more than a year to achieve progeny. Therefore, in order to fasten the breeding processes, the juvenility period needs to be reduced. The juvenility in plants is regulated by set of various flowering genes. The citrus fruit and leaves possess various medicinal properties and are subjected to intensive breeding programs to produce hybrids with improved quality traits. In order to break juvenility in Citrus, it is important to study the role of flowering genes. The present study involved identification of genes regulating flowering in Citrus sinensis L. Osbeck via homology based approach. The structural and functional characterization of these genes would help in targeting genome editing techniques to induce mutations in these genes for producing desirable results. RESULTS: A total of 43 genes were identified which were located on all the 9 chromosomes of citrus. The in-silico analysis was performed to determine the genetic structure, conserved motifs, cis-regulatory elements (CREs) and phylogenetic relationship of the genes. A total of 10 CREs responsible for flowering were detected in 33 genes and 8 conserved motifs were identified in all the genes. The protein structure, protein-protein interaction network and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed to study the functioning of these genes which revealed the involvement of flowering proteins in circadian rhythm pathways. The gene ontology (GO) and gene function analysis was performed to functionally annotate the genes. The structure of the genes and proteins were also compared among other Citrus species to study the evolutionary relationship among them. The expression study revealed the expression of flowering genes in floral buds and ovaries. The qRT-PCR analysis revealed that the flowering genes were highly expressed in bud stage, fully grown flower and early stage of fruit development. CONCLUSIONS: The findings suggested that the flowering genes were highly conserved in citrus species. The qRT-PCR analysis revealed the tissue specific expression of flowering genes (CsFT, CsCO, CsSOC, CsAP, CsSEP and CsLFY) which would help in easy detection and targeting of genes through various forward and reverse genetic approaches.

Elucidating the effect of TiO2 nanoparticles on mung bean rhizobia via in vitro assay: Influence on growth, morphology, and plant growth promoting traits.

Titanium dioxide nanoparticles (TiO2 NPs) are among the most commonly used nanomaterials and are most likely to end up in soil. Therefore, it is pertinent to study the interaction of TiO2 NPs with soil microorganisms. The present in vitro broth study evaluates the impacts of low-dose treatments (0, 1.0, 5.0, 10.0, 20.0, and 40.0 mg L-1 ) of TiO2 NPs on cell viability, morphology, and plant growth promoting (PGP) traits of rhizobia isolated from mung bean root nodule. Two types of TiO2 NPs, that is, mixture of anatase and rutile, and anatase alone were used in the study. These TiO2 NPs were supplemented in broth along with a multifunctional isolate (Bradyrhizobium sp.) and two reference cultures. The exposure of TiO2 (anatase+rutile) NPs at low concentrations (less than 20.0 mg L-1 ) enhanced the cell growth, and total soluble protein content, besides improving the phosphate solubilization, Indole-3-acetic acid (IAA) production, siderophore, and gibberellic acid production. The TiO2 (anatase) NPs enhanced exopolysaccharide (EPS) production by the test rhizobial cultures. The radical scavenging assay was performed to reveal the mode of action of the nano-TiO2 particles. The study revealed higher reactive oxygen species (ROS) generation by the TiO2 (anatase) NPs as compared with TiO2 (anatase+rutile) NPs. Exposure to TiO2 NPs also altered the morphology of rhizobial cells. The findings suggest that TiO2 NPs could act as promoters of PGP traits of PGP bacteria when applied at appropriate lower doses.

In vitro induction and selection of mutants obtained through gamma irradiation with improved processing traits in potato (Solanum tuberosum L.).

PURPOSE: This manuscript aimed for the generation of γ-irradiation derived mutants of potato genotype PAU/RR-1501 possessing desirable processing traits. MATERIALS AND METHODS: Nodal cuttings from virus-free explants were established on basal MS medium and irradiated with different doses (0, 5, 10 and 20 Gy) of γ-irradiation. The 5 and 10 Gy treated plantlets were multiplied and used for micro-tuber induction. Harvested micro-tubers were planted in pots for the selection and evaluation of mutants in M1V2 generation. RESULTS: Four weeks post-treatment, plantlets (5 Gy) showed enhanced growth as compared to the control while 20 Gy treatment exhibited completely ceased shoot growth. The highest number and weight of mini-tubers per plant was recorded for 10 Gy followed by 5 Gy treatment as compared to control. The γ-irradiation treatments caused changes in the skin color and shape of M1V2 tubers. CONCLUSION: Under the 5 Gy treatment 49.9% of clones produced exhibited cream and 8.53% brown skin color. Nine putative mutants were identified in genotype PAU/RR-1501 exhibiting promising processing traits.

Agroinfiltration-based transient genome editing for targeting phytoene desaturase gene in kinnow mandarin (C. reticulata Blanco).

Citrus reticulata Blanco also known as kinnow mandarin is a widely grown horticultural crop in Punjab. CRISPR/Cas9 technology is being widely used for generation of varieties with increased resilience towards abiotic and biotic stresses as well as improved horticultural traits. Xanthomonas citri subsp. citri (Xcc)-mediated Agroinfiltration offers a fast and transgene-free method for the delivery of CRISPR/Cas9 constructs for systemic introduction into plants for functional genomics and expression studies. The technology is currently unexplored in kinnow mandarin. This study is aimed at establishing an efficient method of Cas9 delivery for transient knockout of PDS (phytoene desaturase) gene in kinnow mandarin. The construct pKO-119-PDS N-Cas9/sgRNA:PDS1 carrying sgRNA and Cas9 enzyme was delivered into the dorsal surface of young leaves of kinnow mandarin. The leaves showed albino patches at the point of injection within 60 h. Two surfactants (Triton-X and Silwet™) were used to ease the Agroinfiltration process which resulted in variation in the expression of vector. The Sanger's analysis of the treated plants showed a substitution within the sgRNA region which resulted in change in amino acid from proline to serine. The protocol provides a feasible and an efficient method for genome editing in C. reticulata which could be helpful in future studies aimed at genome editing as well as genetic transformation.

Genetics and marker-assisted breeding for sex expression in cucumber.

Cucumber is an important vegetable crop that provides an accessible draft genome, which has significantly expedited research in various fields of molecular genetics. Cucumber breeders have been employing various methodologies to improve the yield and quality of the crop. These methodologies comprise enhancement of disease resistance, use of gynoecious sex types and their association with parthenocarpy, alterations in plant architecture, and enhancement of genetic variability. The genetics of sex expression are a complex trait in cucumbers but are very significant for the genetic improvement of cucumber crop. This review comprises an explanation of the current status of gene(s) involvement and its expression studies, the inheritance of genes, molecular markers, and genetic engineering associated with sex determination, as well as a discussion of the role of ethylene in sex expression and sex-determining genes of the ACS family. There is no doubt that gynoecy is an important trait among all sex forms of cucumber for heterosis breeding, but if it is associated with parthenocarpy, fruit yield can be enhanced to a greater extent under favorable conditions. However, little information is available with regard to parthenocarpy in gynoecious-type cucumber. This review sheds light on the genetics and molecular mapping of sex expression and could be beneficial especially to cucumber breeders and other scientists working on crop improvement via traditional and molecular assistant approaches.

Genome-wide identification and characterization of parthenocarpic fruit set-related gene homologs in cucumber (Cucumis sativus L.).

Cucumber (Cucumis sativus L.), a major horticultural crop, in the family Cucurbitaceae is grown and consumed globally. Parthenocarpy is an ideal trait for many fruit and vegetables which produces seedless fruit desired by consumers. The seedlessness occurs when fruit develops without fertilization which can be either natural or induced. So far, a limited number of genes regulating parthenocarpic fruit set have been reported in several fruit or vegetable crops, most of which are involved in hormone biosynthesis or signalling. Although parthenocarpic cucumber has been widely used in commercial production for a long time; its genetic basis is not well understood. In this study, we retrieved thirty five parthenocarpy fruit-set related genes (PRGs) from bibliomic data in various plants. Thirty-five PRG homologs were identified in the cucumber genome via homology-based search. An in silico analysis was performed on phylogenetic tree, exon-intron structure, cis-regulatory elements in the promoter region, and conserved domains of their deduced proteins, which provided insights into the genetic make-up of parthenocarpy-related genes in cucumber. Simple sequence repeat (SSR) sequences were mined in these PRGs, and 31 SSR markers were designed. SSR genotyping identified three SSRs in two polymorphic genes. Quantitative real-time PCR of selected genes was conducted in five cucumber lines with varying degrees of parthenocarpic fruit set capacities, which revealed possible association of their expression with parthenocarpy. The results revealed that homologs CsWD40 and CsPIN-4 could be considered potential genes for determination of parthenocarpy as these genes showed parental polymorphism and differential gene expression in case of parthenocarpic and non-parthenocarpic parents.

Agroinfiltration Mediated Scalable Transient Gene Expression in Genome Edited Crop Plants.

Agrobacterium-mediated transformation is one of the most commonly used genetic transformation method that involves transfer of foreign genes into target plants. Agroinfiltration, an Agrobacterium-based transient approach and the breakthrough discovery of CRISPR/Cas9 holds trending stature to perform targeted and efficient genome editing (GE). The predominant feature of agroinfiltration is the abolishment of Transfer-DNA (T-DNA) integration event to ensure fewer biosafety and regulatory issues besides showcasing the capability to perform transcription and translation efficiently, hence providing a large picture through pilot-scale experiment via transient approach. The direct delivery of recombinant agrobacteria through this approach carrying CRISPR/Cas cassette to knockout the expression of the target gene in the intercellular tissue spaces by physical or vacuum infiltration can simplify the targeted site modification. This review aims to provide information on Agrobacterium-mediated transformation and implementation of agroinfiltration with GE to widen the horizon of targeted genome editing before a stable genome editing approach. This will ease the screening of numerous functions of genes in different plant species with wider applicability in future.

Differential Antimycotic and Antioxidant Potentials of Chemically Synthesized Zinc-Based Nanoparticles Derived from Different Reducing/Complexing Agents against Pathogenic Fungi of Maize Crop.

The present study aimed for the synthesis, characterization, and comparative evaluation of anti-oxidant and anti-fungal potentials of zinc-based nanoparticles (ZnNPs) by using different reducing or organic complexing-capping agents. The synthesized ZnNPs exhibited quasi-spherical to hexagonal shapes with average particle sizes ranging from 8 to 210 nm. The UV-Vis spectroscopy of the prepared ZnNPs showed variation in the appearance of characteristic absorption peak(s) for the various reducing/complexing agents i.e., 210 (NaOH and NaBH4), 220 (albumin, and thiourea), 260 and 330 (starch), and 351 nm (cellulose) for wavelengths spanning over 190-800 nm. The FT-IR spectroscopy of the synthesized ZnNPs depicted the functional chemical group diversity. On comparing the antioxidant potential of these ZnNPs, NaOH as reducing agent, (NaOH (RA)) derived ZnNPs presented significantly higher DPPH radical scavenging potential compared to other ZnNPs. The anti-mycotic potential of the ZnNPs as performed through an agar well diffusion assay exhibited variability in the extent of inhibition of the fungal mycelia with maximum inhibition at the highest concentration (40 mg L-1). The NaOH (RA)-derived ZnNPs showcased maximum mycelial inhibition compared to other ZnNPs. Further, incubation of the total genomic DNA with the most effective NaOH (RA)-derived ZnNPs led to intercalation or disintegration of the DNA of all the three fungal pathogens of maize with maximum DNA degrading effect on Macrophomina phaseolina genomic DNA. This study thus identified that differences in size and surface functionalization with the protein (albumin)/polysaccharides (starch, cellulose) diminishes the anti-oxidant and anti-mycotic potential of the generated ZnNPs. However, the NaOH emerged as the best reducing agent for the generation of uniform nano-scale ZnNPs which possessed comparably greater anti-oxidant and antimycotic activities against the three test maize pathogenic fungal cultures.

Function of the blood-brain barrier and restriction of drug delivery to invasive glioma cells: findings in an orthotopic rat xenograft model of glioma.

Despite aggressive treatment with radiation and chemotherapy, recurrence of glioblastoma multiforme (GBM) is inevitable. The objective of this study was to show that the blood-brain barrier (BBB), through a combination of tight junctions and active efflux transporters in the brain microvasculature, can significantly restrict delivery of molecularly targeted agents to invasive glioma cells. Transgenic mice lacking P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) were used to study efflux of erlotinib at the BBB. A U87 rat xenograft model of GBM was used to investigate the regional distribution of erlotinib to the tumor, and brain regions surrounding the tumor. The effect of concurrent administration of elacridar on regional tumor distribution of erlotinib was evaluated. We show that erlotinib transport across an intact BBB is significantly restricted due to P-gp- and Bcrp-mediated efflux transport. We then show that the BBB is sufficiently intact in areas of brain adjacent to the tumor core to significantly restrict erlotinib delivery. Inhibition of P-gp and Bcrp by the dual inhibitor elacridar dramatically increased erlotinib delivery to the tumor core, rim, and normal brain. These results provide conclusive evidence of the impact that active efflux at the BBB has on the delivery of molecularly targeted therapy to different tumor regions in glioma. These data also support the possibility that the repeated failure of clinical trials of new drugs for gliomas may be in part due to a failure to achieve effective concentrations in invasive tumor cells that reside behind an intact BBB.

A novel approach to evaluate the immunogenicity of viral antigens of clinical importance in HLA transgenic murine models.

Transgenic (Tg) mice expressing HLA class I alleles and lacking murine MHC class I represent a useful model for the pre-clinical evaluation of human vaccines, which focus on induction of CD8(+) T-cell responses. We have developed a platform to be used in Tg mice for exploring the immunogenicity of T-cell targets, whose immunologic epitopes have yet to be defined. To test the attributes of the evaluation system in the context of an important human pathogen, we have explored multiple antigens from cytomegalovirus (CMV). A panel of recombinant modified vaccinia Ankara (MVA) vectors, expressing various CMV proteins (CMV-MVA) was used to immunize HLA-A*0201, B*0702 and A*1101 Tg mice. Immune splenocytes were in vitro stimulated (IVS) either using syngeneic lipo-polysaccharide activated lymphoblasts or Tg HLA-I matched human EBV-transformed B-lymphoblastoid cells (LCL), both loaded with peptide libraries, encompassing the CMV protein under investigation. IVS performed with peptide library loaded lymphoblasts failed to provide a reliable stimulation. In contrast, the usage of LCL as antigen presenting cells (APC) of CMV peptide libraries resulted in a consistent and specific amplification of the Tg T-cell response in animals immunized with CMV-MVAs. The LCL IVS method reliably allowed defining the immunogenicity and immunodominant CD8(+) T-cell regions of uncharacterized CMV antigens. The combination of CMV-MVA vectors, unbiased pools of CMV-specific peptide libraries presented by Tg HLA-I matched LCL constitutes a valid tool for the pre-clinical evaluation of model candidate vaccines. This convenient method could find application to investigate the immunogenicity profile of cancer antigens or proteins from infectious human pathogens.

Programmed death-1 expression in liver transplant recipients as a prognostic indicator of cytomegalovirus disease.

Immunological parameters that distinguish solid-organ transplant (SOT) recipients at risk for life-threatening cytomegalovirus (CMV) disease are being actively pursued to aid posttransplant management. A candidate marker is programmed death (PD)-1 receptor, whose overexpression has been associated with disease progression during persistent viral infections. To determine whether levels of this negative regulator of T cell activity are altered in SOT recipients with symptoms of CMV disease, a comparative PD-1 expression analysis was done in healthy, CMV-positive individuals and in liver transplant recipients. PD-1 levels were measured among the total population of CD8(+) and CD8(+) T cells binding to CMV-specific major histocompatibility complex class I tetramers. Minimal PD-1 expression was found in the healthy, CMV-positive cohort, and symptomatic SOT recipients had significantly higher PD-1 levels. PD-1 up-regulation was significantly associated with incipient and overt CMV disease and with viremia. Our findings suggest that PD-1 could be developed as a prognostic tool to predict CMV disease and guide therapeutic interventions.