Morpho-histology of co-occurrence of somatic embryos, shoots, and inflorescences within a callus of Limonium 'Misty Blue'.

This is the first attempt to report the co-occurrence of somatic embryos, shoots, and inflorescences and their sequential development from stem cell niches of an individual callus mass through morpho-histological study of any angiosperm. In the presence of a proper auxin/cytokinin combination, precambial stem cells from the middle layer of a compact callus, which was derived from the thin cell layer of the inflorescence rachis of Limonium, expressed the highest level of totipotency and pluripotency and simultaneously developed somatic embryos, shoots, and inflorescences. This study also proposed the concept of programmed cell death during bipolar somatic embryo and unipolar shoot bud pattern formation. The unique feature of this research was the stepwise histological description of in vitro racemose inflorescence development. Remarkably, during the initiation of inflorescence development, either a unipolar structure with open vascular elements or an independent bipolar structure with closed vascular elements were observed. The protocol predicted the production of 6.6 ± 0.24 and 7.4 ± 0.24 somatic embryos and shoots, respectively, from 400 mg of callus, which again multiplied, rooted, and acclimatised. The plants' ploidy level and genetic fidelity were assessed randomly before acclimatisation by flow cytometry and inter simple sequence repeats (ISSR) marker analysis. Finally, the survivability and flower quality of the regenerated plants were evaluated in the field.

microRNAomic profiling of maize root reveals multifaceted mechanisms to cope with Cr (VI) stress.

Chromium (Cr) contamination of soil and water poses serious threats to agricultural crop production. MicroRNAs (miRNAs) are conserved, non-coding small RNAs that play pivotal roles in plant growth, development and stress responses through fine-tuning of post-transcriptional gene expression. To better understand the molecular circuit of Cr-responsive miRNAs, two sRNA libraries were prepared from control and Cr (VI) [100 ppm] exposed maize roots. Using deep sequencing, we identified 80 known (1 up and 79 down) and 18 downregulated novel miRNAs from Cr (VI) challenged roots. Gene ontology (GO) analysis reveals that predicted target genes of Cr (VI) responsive miRNAs are potentially involved in diverse cellular and biological processes including plant growth and development (miR159c, miR164d, miR319b-3p and zma_25.145), redox homeostasis (miR528-5p, miR396a-5p and zma_9.132), heavy metal uptake and detoxification (miR159f-5p, 164e-5p, miR408a, miR444f and zma_2.127), signal transduction (miR159f, miR160a-5p, miR393a-5p, miR408-5p and zma_43.158), cell signalling (miR156j, 159c-5p, miR166c-5p and miR398b). Higher accumulation of Cr in maize roots might be due to upregulation of ABC transporter G family member 29 targeted by miR444f. Instead of isolated increase in SOD expression, significant decline in GSH:GSSH ratio and histochemical staining strongly suggest Cr (VI) stress mediated disruption of ROS scavenging machinery thus unbalancing normal cellular homeostasis. Moreover, miR159c-mediated enhanced expression of GAMYB might be a reason for impaired root growth under Cr (VI) stress. In a nutshell, the present microRNAomic study sheds light on the miRNA-target gene regulatory network involved in adaptive responses of maize seedlings to Cr (VI) stress.

Impact of CuO nanoparticles on maize: Comparison with CuO bulk particles with special reference to oxidative stress damages and antioxidant defense status.

The present study aimed to systematically investigate the particle size effects of copper (II) oxide [CuO nanoparticles (<50 nm) and CuO bulk particles (<10 µm)] on maize (Zea mays L.). Bioaccumulation of Cu, in vivo ROS generation, membrane damage, transcriptional modulation of antioxidant genes, cellular redox status of glutathione and ascorbate pool, expression patterns of COPPER TRANSPORTER 4 and stress responsive miRNAs (miR398a, miR171b, miR159f-3p) with their targets were investigated for better understanding of the underlying mechanisms and the extent of CuO nanoparticles and CuO bulk particles induced oxidative stress damages. More restricted seedling growth, comparatively higher membrane injury, marked decline in the levels of chlorophylls and carotenoids and severe oxidative burst were evident in CuO bulk particles challenged leaves. Dihydroethidium and CM-H2DCFDA staining further supported elevated reactive oxygen species generation in CuO bulk particles stressed roots. CuO bulk particles exposed seedlings accumulated much higher amount of Cu in roots as compared to CuO nanoparticles stressed plants with low root-to-shoot Cu translocation. Moderately high GR expression with maintenance of a steady GSH-GSSG ratio in CuO nanoparticles challenged leaves might be accountable for their rather improved performance under stressed condition. miR171b-mediated enhanced expression of SCARECROW 6 might participate in the marked decline of chlorophyll content in CuO bulk particles exposed leaves. Ineffective recycling of AsA pool is another decisive feature of inadequate performance of CuO bulk particles stressed seedlings in combating oxidative stress damages. Taken together, our findings revealed that toxicity of CuO bulk particles was higher than CuO nanoparticles and the adverse effects of CuO bulk particles on maize seedlings might be due to higher Cu ions dissolution.

Assessment of ZnO-NPs toxicity in maize: An integrative microRNAomic approach.

Rapid expansion of nanotechnology and indiscriminate discharge of metal oxide nanoparticles (NPs) into the environment pose a serious hazard to the ecological receptors including plants. To better understand the role of miRNAs in ZnO-NPs stress adaptation, two small RNA libraries were prepared from control and ZnO-NPs (800 ppm, <50 nm particle size) stressed maize leaves. Meager performance of ZnO-NPs treated seedlings was associated with elevated tissue zinc accumulation, enhanced ROS generation, loss of root cell viability, increased foliar MDA content, decrease in chlorophyll and carotenoids contents. Deep sequencing identified 3 (2 known and 1 novel) up- and 77 (73 known and 4 novel) down-regulated miRNAs from ZnO-NPs challenged leaves. GO analysis reveals that potential targets of ZnO-NPs responsive miRNAs regulate diverse biological processes viz. plant growth and development (miR159f-3p, zma_18), ROS homeostasis (miR156b, miR166l), heavy metal transport and detoxification (miR444a, miR167c-3p), photosynthesis (miR171b) etc. Up-regulation of SCARECROW 6 in ZnO-NPs treated leaves might be responsible for suppression of chlorophyll biosynthesis leading to yellowing of leaves. miR156b.1 mediated up-regulation of CALLOSE SYNTHASE also does not give much protection against ZnO-NPs treatment. Taken together, the findings shed light on the miRNA-guided stress regulatory networks involved in plant adaptive responses to ZnO-NPs stress.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis.

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26µM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65µM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32µM KIN and 2.22µM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1.

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62 ± 0.01), with average length 4.19 ± 0.01 cm, was initially achieved with medium containing 0.5 mg/l N(6)-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0 mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies.

Identification and validation of a new male sex-specific ISSR marker in pointed gourd (Trichosanthes dioica Roxb.).

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.