Comparative Evaluation of Preformed Stainless Steel Crown's Crazing at Different Autoclave Cycles.

Aim: The study aimed to assess and compare the crazing of preformed stainless steel (SS) crowns at various autoclave cycles. Materials and methods: Around 15 SS crowns were subjected to zero (unsterilized), one, three, five, and seven autoclave cycles at 121°C, 15 psi for 20 minutes. After each autoclave cycle, the mesial surface of each crown from occlusal to cervical portions was examined under a 40× stereomicroscope. Crazing was evaluated using Wickersham's criteria. Results: One autoclave cycle does not produce any cracks, whereas the higher the number of autoclave cycles, the higher the cracks, which affects the prognosis of the SS crown treatment. Conclusion: Surface alterations of SS crowns after different autoclave cycles were significant. So, preformed SS crowns should be discarded after sterilizing once or twice during clinical practice. How to cite this article: Sowkarthicaa P, Mathian VM, Gawthaman M, et al. Comparative Evaluation of Preformed Stainless Steel Crown's Crazing at Different Autoclave Cycles. Int J Clin Pediatr Dent 2023;16(S-1):S77-S80.

Transgenic Rice Expressing Isoflavone Synthase Gene from Soybean Shows Resistance Against Blast Fungus (Magnaporthe oryzae).

The isoflavones are a group of plant secondary metabolites primarily synthesized in legumes and are known for their role in improving human health and plant disease resistance. The isoflavones, especially genistein, act as precursors for the production of phytoalexins, which may induce broad-spectrum disease resistance in plants. In this study, we screened transgenic rice lines expressing the isoflavone synthase (GmIFS1) gene from soybean for rice blast (Magnaporthe oryzae) resistance. Two homozygous transgenic lines (I2 and I10), based on single copy gene integration, were identified. The expression of GmIFS1 in transgenic lines was confirmed by quantitative real-time PCR. Genistein was detected in the transgenic lines using liquid chromatography with tandem mass spectrometry. Subsequently, the transgenic lines were evaluated against the rice blast pathogen, isolate YJ54 (race IB-54). The results indicated that >60% of the plants in both the lines (I2 and I10) showed resistance against the blast pathogen. The progenies of one of the resistant transgenic lines (I10) also showed >65% resistance against rice blast. The resistance of these transgenic lines against rice blast may be attributed to the synthesis of isoflavone (genistein) in rice.

Dual-targeting by CRISPR/Cas9 leads to efficient point mutagenesis but only rare targeted deletions in the rice genome.

The present study investigated the efficiency of CRISPR/Cas9 in creating genomic deletions as the basis of its application in removing selection marker genes or the intergenic regions. Three loci, representing a transgene and two rice genes, were targeted at two sites each, in separate experiments, and the deletion of the defined fragments was investigated by PCR and sequencing. Genomic deletions were found at a low rate among the transformed callus lines that could be isolated, cultured, and regenerated into plants harboring the deletion. However, randomly regenerated plants showed mixed genomic effects, and generally did not harbor heritable genomic deletions. To determine whether point mutations occurred at each targeted site, a total of 114 plants consisting of primary transgenic lines and their progeny were analyzed. Ninety-three plants showed targeting, 60 of which were targeted at both sites. The presence of point mutations at both sites was correlated with the guide RNA efficiency. In summary, genomic deletions through dual-targeting by the paired-guide RNAs were generally observed in callus, while de novo point mutations at one or both sites occurred at high rates in transgenic plants and their progeny, generating a variety of insertion-deletions or single-nucleotide variations. In this study, point mutations were exceedingly favored over genomic deletions; therefore, for the recovery of plant lines harboring targeted deletions, identifying early transformed clones harboring the deletions, and isolating them for plant regeneration is recommended.

Comparative analysis of the root transcriptomes of cultivated sweetpotato (Ipomoea batatas [L.] Lam) and its wild ancestor (Ipomoea trifida [Kunth] G. Don).

BACKGROUND: The complex process of formation of storage roots (SRs) from adventitious roots affects sweetpotato yield. Identifying the genes that are uniquely expressed in the SR forming cultivated species, Ipomoea batatas (Ib), and its immediate ancestral species, Ipomoea trifida (It), which does not form SRs, may provide insights into the molecular mechanisms underlying SR formation in sweetpotato. RESULTS: Illumina paired-end sequencing generated ~208 and ~200 million reads for Ib and It, respectively. Trinity assembly of the reads resulted in 98,317 transcripts for Ib and 275,044 for It, after post-assembly removal of trans-chimeras. From these sequences, we identified 4,865 orthologous genes in both Ib and It, 60 paralogous genes in Ib and 2,286 paralogous genes in It. Among paralogous gene sets, transcripts encoding the transcription factor RKD, which may have a role in nitrogen regulation and starch formation, and rhamnogalacturonate lyase (RGL) family proteins, which produce the precursors of cell wall polysaccharides, were found only in Ib. In addition, transcripts encoding a K+ efflux antiporter (KEA5) and the ERECTA protein kinase, which function in phytohormonal regulation and root proliferation, respectively, were also found only in Ib. qRT-PCR indicated that starch and sucrose metabolism genes, such as those encoding ADP-glucose pyrophosphorylase and beta-amylase, showed lower expression in It than Ib, whereas lignin genes such as caffeoyl-CoA O-methyltransferase (CoMT) and cinnamyl alcohol dehydrogenase (CAD) showed higher expression in It than Ib. A total of 7,067 and 9,650 unique microsatellite markers, 1,037,396 and 495,931 single nucleotide polymorphisms (SNPs) and 103,439 and 69,194 InDels in Ib and It, respectively, were also identified from this study. CONCLUSION: The detection of genes involved in the biosynthesis of RGL family proteins, the transcription factor RKD, and genes encoding a K+ efflux antiporter (KEA5) and the ERECTA protein kinase only in I. batatas indicate that these genes may have important functions in SR formation in sweetpotato. Potential molecular markers (SNPs, simple sequence repeats and InDels) and sequences identified in this study may represent a valuable resource for sweetpotato gene annotation and may serve as important tools for improving SR formation in sweetpotato through breeding.

Gene stacking in plant cell using recombinases for gene integration and nucleases for marker gene deletion.

BACKGROUND: Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts. Recombinases such as Cre-lox, on the other hand, efficiently drive site-specific integrations in plants. However, the reversibility of Cre-lox recombination, due to the incorporation of two cis-positioned lox sites, presents a major bottleneck in its application in gene stacking. Here, we describe a strategy of resolving this bottleneck through excision of one of the cis-positioned lox, embedded in the marker gene, by nuclease activity. METHODS: All transgenic lines were developed by particle bombardment of rice callus with plasmid constructs. Standard molecular approach was used for building the constructs. Transgene loci were analyzed by PCR, Southern hybridization, and DNA sequencing. RESULTS: We developed a highly efficient gene stacking method by utilizing powerful recombinases such as Cre-lox and FLP-FRT, for site-specific gene integrations, and nucleases for marker gene excisions. We generated Cre-mediated site-specific integration locus in rice and showed excision of marker gene by I-SceI at ~20 % efficiency, seamlessly connecting genes in the locus. Next, we showed ZFN could be used for marker excision, and the locus can be targeted again by recombinases. Hence, we extended the power of recombinases to gene stacking application in plants. Finally, we show that heat-inducible I-SceI is also suitable for marker excision, and therefore could serve as an important tool in streamlining this gene stacking platform. CONCLUSIONS: A practical approach for gene stacking in plant cell was developed that allows targeted gene insertions through rounds of transformation, a method needed for introducing new traits into transgenic lines for their rapid deployment in the field. By using Cre-lox, a powerful site-specific recombination system, this method greatly improves gene stacking efficiency, and through the application of nucleases develops marker-free, seamless stack of genes at pre-determined chromosomal sites.

Genome-Wide Profiling of Histone Modifications (H3K9me2 and H4K12ac) and Gene Expression in Rust (Uromyces appendiculatus) Inoculated Common Bean (Phaseolus vulgaris L.).

Histone modifications such as methylation and acetylation play a significant role in controlling gene expression in unstressed and stressed plants. Genome-wide analysis of such stress-responsive modifications and genes in non-model crops is limited. We report the genome-wide profiling of histone methylation (H3K9me2) and acetylation (H4K12ac) in common bean (Phaseolus vulgaris L.) under rust (Uromyces appendiculatus) stress using two high-throughput approaches, chromatin immunoprecipitation sequencing (ChIP-Seq) and RNA sequencing (RNA-Seq). ChIP-Seq analysis revealed 1,235 and 556 histone methylation and acetylation responsive genes from common bean leaves treated with the rust pathogen at 0, 12 and 84 hour-after-inoculation (hai), while RNA-Seq analysis identified 145 and 1,763 genes differentially expressed between mock-inoculated and inoculated plants. The combined ChIP-Seq and RNA-Seq analyses identified some key defense responsive genes (calmodulin, cytochrome p450, chitinase, DNA Pol II, and LRR) and transcription factors (WRKY, bZIP, MYB, HSFB3, GRAS, NAC, and NMRA) in bean-rust interaction. Differential methylation and acetylation affected a large proportion of stress-responsive genes including resistant (R) proteins, detoxifying enzymes, and genes involved in ion flux and cell death. The genes identified were functionally classified using Gene Ontology (GO) and EuKaryotic Orthologous Groups (KOGs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis identified a putative pathway with ten key genes involved in plant-pathogen interactions. This first report of an integrated analysis of histone modifications and gene expression involved in the bean-rust interaction as reported here provides a comprehensive resource for other epigenomic regulation studies in non-model species under stress.

Co-bombardment, integration and expression of rice chitinase and thaumatin-like protein genes in barley (Hordeum vulgare cv. Conlon).

Pathogenesis-related (PR) proteins associated with degradation of structural components of pathogenic filamentous fungi were overexpressed in the two-rowed malting barley (Hordeum vulgare L.) cultivar Conlon. Transgenes were introduced by co-bombardment with two plasmids, one carrying a rice (Oryza sativa L.) chitinase gene (chi11) and another carrying a rice thaumatin-like protein gene (tlp). Each gene was under the control of the maize ubiquitin (Ubi1) promoter. Fifty-eight primary transformants from three independent transformation events were regenerated. T(1) plants with high rice chi11 and tlp protein expression levels were advanced to identify T(2) homozygotes by herbicide spray and subjected to further molecular analyses. T(3) progeny from one event (E2) had stable integration and expression of the rice chi11 and tlp while those from the other events (E1 and E3) showed stable integration only of tlp. The successful production of these lines overexpressing the antifungal chi and tlp proteins provides materials to test the effects of these genes on a variety of fungal diseases that attack barley and to serve as potential additional sources of disease resistance.