Plant Biotechnology and Molecular Biology

A B S T R A C T Exosomes are biological nanovesicles that are involved in cell–cell communication via the functionally-active cargo (such as miRNA, mRNA, DNA and proteins). Because of their nanosize, exosomes are explored as nanodevices for the development of new therapeutic applications. However, bulk, safe and cost-effective production of exosomes is not available. Here, we show that bovine milk can serve as a scalable source of exosomes that can act as a carrier for chemotherapeutic/chemopreventive agents. Drug-loaded exosomes showed significantly higher efficacy compared to free drug in cell culture studies and against lung tumor xenografts in vivo. Moreover, tumor targeting ligands such as folate increased cancer-cell targeting of the exosomes resulting in enhanced tumor reduction. Milk exosomes exhibited cross-species tolerance with no adverse immune and inflammatory response. Thus, we show the versatility of milk exosomes with respect to the cargo it can carry and ability to achieve tumor targetability. This is the first report to identify a biocompatible and cost-effective means of exosomes to enhance oral bioavailability, improve efficacy and safety of drugs.

Endophytic microbes, since their discovery are known to live asymptomatically inside their host throughout different stages of their life cycle and play crucial role in growth, development, fitness, and diversification of plant. The plant-endophyte association ranges from mutualism to pathogenicity. Endophytic microbes help the host to combat diverse arrays of both biotic and abiotic stressful conditions. Endophytic microbes play a major role in growth enhancement of their host, solubilization of macronutrients such as phosphorous, potassium, and zinc, fixation of atmospheric nitrogen, synthesis of phytohormones, siderophores, hydrogen cyanide, ammonia, and act as a biocontrol agent against wide array of phytopathogens etc. Endophytic microbes are beneficial to plant by directly promoting their growth or indirectly by inhibiting the growth of phytopathogens. Over a long period of co-evolution endophytic microbes have attained the mechanism of synthesis of various hydrolytic enzymes such as pectinase, xylanases, cellulase, proteinase etc. which promote the penetration of endophytic microbes into tissues of plants. The enzymes are very specific in their action on substrate and play different roles, such as convert macromolecule into small one as well as toxic substance into less toxic. The effective usages of endophytic microbes in the form of biofertilizers reduce the usage of chemical fertilizers. Endophytic microbes belong to different phyla such as Actinobacteria, Acidobacteria, Bacteroidetes, Deinococcus-thermus, Firmicutes, Proteobacteria, and Verrucomicrobia. The most predominant and studied endophytic bacteria belonged to Proteobacteria followed by Firmicutes and then by Actinobacteria. The most dominant among reported genera in most of the leguminous and non-leguminous plants are Bacillus, Pseudomonas, Fusarium, Burkholderia, Rhizobium, and Klebsiella. In future, for the sustainable agriculture, endophytic microbes have a wide range of potential for maintaining health of plant as well as environmental conditions. The present review is focused on the research being done on endophytic microbes including their diversity in leguminous as well as non-leguminous crops, biotechnological applications, and ability to promote the growth of plant

Modern genome editing (GE) techniques, which include clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system, transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs) and LAGLIDADG homing endonucleases (meganucleases), have so far been used for engineering disease resistance in crops. The use of GE technologies has grown very rapidly in recent years with numerous examples of targeted mutagenesis in crop plants, including gene knockouts, knockdowns, modifications, and the repression and activation of target genes. CRISPR/Cas9 supersedes all other GE techniques including TALENs and ZFNs for editing genes owing to its unprecedented efficiency, relative simplicity and low risk of off-target effects. Broad-spectrum disease resistance has been engineered in crops by GE of either specific host-susceptibility genes (S gene approach), or cleaving DNA of phytopathogens (bacteria, virus or fungi) to inhibit their proliferation. This review focuses on different GE techniques that can potentially be used to boost molecular immunity and resistance against different phytopathogens in crops, ultimately leading to the development of promising disease-resistant crop varieties.

Maize is the principal grain crop of the world. It is also the crop where genetic engineering has been employed to a great extent to improve its various traits. The ability to transform maize is a crucial step for application of gene technology in maize improvement. There have been constant improvements in the maize transformation technologies over past several years. The choice of genotype and the explant material to initiate transformation and the different types of media to be used in various stages of tissue culture can have significant impact on the outcomes of the transformation efforts. Various methods of gene transfer, like the particle bombardment, protoplast transformation, Agrobacterium-mediated, in planta transformation, etc., have been tried and improved over years. Similarly, various selection systems for retrieval of the transformants have been attempted. The commercial success of maize transformation and transgenic development is unmatched by any other crop so far. Maize transformation with newer gene editing technologies is opening up a fresh dimension in transformation protocols and work-flows. This review captures the various past and recent facets in improvement in maize transformation technologies and attempts to present a comprehensive updated picture of the current state of the art in this area.

Small RNAs including micro RNAs (miRNA) play an indispensable role in cell signaling mechanisms. Generally, miRNAs that are 20–24 nucleotides long bind to specific
complementary transcripts, attenuating gene expression at the post-transcriptional level or via translational inhibition. In plants, miRNAs have emerged as the principal regulator
of various stress responses, including low nutrient availability. It has been reported that miRNAs are vital for maintaining nutrient homeostasis in plants by regulating the
expression of transporters that are involved in nutrient uptake and mobilization. The present review highlights the role of various miRNAs in several macro- or micronutrient
deficiencies in plants. Understanding the regulation of different transporters by miRNAs will aid in elucidating the underlying molecular signal transduction mechanisms during nutritional stress. Recent findings regarding nutrient related-miRNAs and their gene regulation machinery may delineate a novel platform for improving the nutritional status of cereal grains or crop biofortification programs in the future.

Analysis of time series of images can quantify plant growth and development, including the effects of genetic mutations (phenotypes) that give information about gene function. Here is demonstrated a software application named HYPOTrace that automatically extracts growth and shape information from electronic gray-scale images of Arabidopsis (Arabidopsis thaliana) seedlings. Key to the method is the iterative application of adaptive local principal components analysis to extract a set of ordered midline points (medial axis) from images of the seedling hypocotyl. Pixel intensity is weighted to avoid the medial axis being diverted by the cotyledons in areas where the two come in contact. An intensity feature useful for terminating the midline at the hypocotyl apex was isolated in each image by subtracting the baseline with a robust local regression algorithm. Applying the algorithm to time series of images of Arabidopsis seedlings responding to light resulted in automatic quantification of hypocotyl growth rate, apical hook opening, and phototropic bending with high spatiotemporal resolution. These functions are demonstrated here on wild-type, cryptochrome1, and phototropin1 seedlings for the purpose of showing that HYPOTrace generated expected results and to show how much richer the machine-vision description is compared to methods more typical in plant biology. HYPOTrace is expected to benefit seedling development research, particularly in the photomorphogenesis field, by replacing many tedious, error-prone manual measurements with a precise, largely automated computational tool.

In Narcissus tazetta, a monocotyledonous bulbous geophyte, floral initiation and differentiation occur within the bulb during the quiescent period in summer, when ambient temperatures are relatively high and the bulb is located underground with no foliage or roots. In many plant species, FLOWERING LOCUS T (FT) and its homologues are considered powerful promoters of flowering. The Narcissus FT gene homologue (NtFT) was isolated, and organ-specific expression patterns of NtFT during the annual cycle and reproductive development under different temperature regimes were analysed using quantitative reverse transcription–PCR (qRT–PCR) and RNA in situ hybridization. During floral induction, NtFT was not expressed in bulb scales, roots, or foliage leaves, but it was detected inside the bulb in the apical meristem and leaf primordia. The expression of another key flowering gene, NLF, the LEAFY homologue in N. tazetta, was also observed only in meristem and leaf primordia within the bulbs; however, its expression did not coincide with that of NtFT during meristem transition to reproductive stage. Under high temperatures (25–30 °C) in the dark, NtFT expression occurred simultaneously with floral induction timing, indicating that floral induction is affected by high temperatures but not by photoperiod or vernalization. Monitoring the apical meristem of Narcissus in February–August of two growing seasons under ambient and controlled storage conditions showed that transition to flowering is temperature dependent and varies between years. Lack of NtFT and NLF expression in foliage leaves suggests that flower initiation control in Narcissus differs from that in common model plants

Halophyte is a distinctive group of plants that can survive, even well flourish, at a concentration of Na + and Cl − ions along with heavy metals that would be lethal to most of the agricultural crop species. These capabilities make certain halophytes good contenders for phytoremediation through phytoextraction or phytostabilization of the salt and heavy metals (HMs) in polluted soils. Thus, the present study elucidates the phytoextraction capacity of three halophytes (Suaeda monoica, Tamarix indica and Cressa critica) growing in saline soil (EC 112 ds m −1), with higher level of HMs rather than a cultivated soil. The accumulation of ions in above-ground tissue was determined in the all three studied plants, considering the fact that maintaining a stable cytosolic Na + /K + ratio has become a crucial salinity tolerance mechanism. The higher salinity of soil resulted in high level of Na + ions in leaves, increased synthesis of osmolyte components and robust antioxidant activities to combat the oxidative stress. As whole, changes in cellular metabolites were determined by using FT-IR spectroscopy, evident as differential FT-IR profiles in both leaves and stem specific to these metabolites. The considerable amounts of HMs accumulation including Zn, Fe, Mn, Cu, Cr, and Cd with highest being Fe in above-ground tissue of all three studied halophytes were obtained. These preliminary findings represent S. monoica, T. indica and C. cretica as potent phytoremedia-tion plant using phytosequestration to accumulate HMs. The present study project a light on the use of these three plants in reclamation of degraded saline soils.

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-b-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.

Gene disruption frequently produces no phenotype in the model plant Arabidopsis thaliana, complicating studies of gene function. Functional redundancy between gene family members is one common explanation but inadequate detection methods could also be responsible. Here, newly developed methods for automated capture and processing of times series of images, followed by computational analysis employing modified linear discriminant analysis (LDA) and wavelet-based differentiation were employed in a study of mutants lacking the Glutamate Receptor-Like 3.3 gene. Root