Acclimatization of drought tolerance with Somaclonal variants of sugarcane (Saccharum officinarum L.)

Background: Sugarcane is basically a water loving crop and scarcity of water is not only affecting its overall production but its total sugar contents as well. Somaclonal variation is an intriguing phenomenon to study various genetic and cellular mechanism under in vitro conditions. The induction of somaclonal variation generates useful variability without sexual reproduction in sugarcane.Methods: CPF-248, a drought sensitive variety was used to induce stress tolerance using somaclonal variation approach. Various combinations of callogenesis and regeneration media were used to induce soma clonal variations. The plants, thus achieved, were subjected to drought selection pressure using different concentrations of PEG, the selected plants were referred as in vitro selected putative somaclonal variants 1 & 2 (IPSV1 & IPSV2). These two plants were then subjected to acclimatize under greenhouse conditions by planting them in soil filled plastic pots. The in vitro grown seedlings named as in vitro selected putative somaclonal variants along with parent plants were subjected to drought conditions by withholding watering for 8 days. After 10 days of treatment, data for photosynthetic and biochemical attributes were recorded and subjected to comparison. The activity of antioxidants enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), peroxidase (POD), ascorbate and carotenoid contents was analyzed using standard protocols.Results: A significant increase in chlorophyll a, b and carotenoid contents were recorded in IPSV1 and IPSV2 compared to parent genotype. Antioxidant enzymes (SOD, APX, CAT, POD) also increased significantly in somaclonal variants and displayed tolerance under drought condition. Moreover, osmotic adjustment was observed in the form of higher rate of total free amino acid and proline contents in both somaclonal variants.Conclusion: The finding suggests that sugarcane (CPF-248) somaclones generated through tissue culture approach are found to be drought tolerant with improved photosynthesis and antioxidant response. Further, somaclonal variant IPSVI, exhibited better response than other variant (IPSV2).Keywords: Drought; Antioxidants; Osmoprotectants; Somaclones; Sugarcan

Phylogenomics resolves the etiology of dieback disease and deciphers Ceratocystis dalbergicans sp.nov., causal agent of Dalbergia sissoo decline

Dalbergia sissoo is one of the most economically important trees in forestry, agroforestry, and horticulture. This tree species is severely threatened by dieback. Widespread dieback outbreaks and infestations have drastically destroyed billions of D. sissoo trees. Hence, we attempted to resolve the dieback etiology through phylogenomics associated with D. sissoo mortality. The Ceratocystis species was evaluated using morphologically investigated fungal isolates collected from dieback-affected tissue plants. Based on the symptomatology, we have differentiated dieback from Fusarium wilt and concluded that the Ceratocystis fimbriata sensu lato complex is causing shisham dieback in Pakistan. As the Ceratocystis species complex is a cryptic species complex, we used genomics and phylogenetic analysis for deciphering its evolutionary hierarchical order. The pathogen’s operational taxonomy was unlocked with the help of phylogenomics, and it was discovered that isolates from D. sissoo represent a species distinct from the other species in the C. fimbriata sensu lato species complex. The name Ceratocystis dalbergicans sp. nov. has been given to the fungus causing dieback disease in D. sissoo

Integrative Pathogenicity Assay and Operational Taxonomy-Based Detection of New Forma Specialis of <i>Fusarium oxysporum</i> Causing Datepalm Wilt

Pathogenicity-associated genes are highly host-specific and contribute to host-specific virulence. We tailored the traditional Koch’s postulates with integrative omics by hypothesizing that the effector genes associated with host-pathogenicity are determinant markers for virulence, and developed Integrative Pathogenicity (IP) postulates for authenticated pathogenicity testing in plants. To set the criteria, we experimented on datepalm (Phoenix dactylifera) for the vascular wilt pathogen and confirmed the pathogen based on secreted in xylem genes (effectors genes) using genomic and transcriptomic approaches, and found it a reliable solution when pathogenicity is in question. The genic regions ITS, TEF1-α, and RPBII of Fusarium isolates were examined by phylogenetic analysis to unveil the validated operational taxonomy at the species level. The hierarchical tree generated through phylogenetic analysis declared the fungal pathogen as Fusarium oxysporum. Moreover, the Fusarium isolates were investigated at the subspecies level by probing the IGS, TEF1-α, and Pgx4 genic regions to detect the forma specialis of F. oxysporum that causes wilt in datepalm. The phylogram revealed a new forma specialis in F. oxysporum that causes vascular wilt in datepalm

In vitro Regeneration of Dalbergia sissoo Roxb. and the Potential for Genetic Transformation

Dalbergia sissoo Roxb. ex DC. (Sissoo) is a native forest tree species in Pakistan. Many ecological and economical uses are associated with this premier timber species, but dieback disease is of major concern. The objective of this study was to develop a protocol for in vitro regeneration of Sissoo that could serve as target material for genetic transformation, in order to improve this species. Callus formation and plantlet regeneration was achieved by culturing cotyledons, immature seeds, and mature embryos on a modified Murashige and Skoog (1962) (MS) medium supplemented with plant growth regulators. Callus induction medium containing 2.71 ?M 2, 4-dichlorophenoxyacetic acid (2,4-D) and 0.93 ?M kinetin produced better callus on all explants tested compared to other treatments, such as 8.88 ?M 6-benzylaminopurine (BA) and 2.69 ?M ?-naphthalene acetic acid (NAA), or 2.71 ?M 2, 4-D and 2.69 ?M NAA. Shoot regeneration was best on MS medium containing 1.4 ?M NAA and 8.88 ?M BA compared to other treatments, such as 1.4 ?M NAA and 9.9 ?M kinetin, or 2.86 ?M indole-3-acetic acid and 8.88 ?M BA. Murashige and Skoog medium containing 1.4 NAA ?M and 8.88 ?M BA was better in general for regeneration regardless of callus induction medium and the type of explant used. Rooting was best on half-strength MS medium with 7.35 ?M indole-3-butyric acid. Regenerated plantlets were acclimatized for plantation in the field. Preliminary genetic transformation potential of D. sissoo was evaluated by particle bombardment of callus explants with a pUbiGus vector. The bombarded tissue showed transient Gus activity 1week after bombardment. Transformation of this woody tree is possible provided excellent regeneration protocols. The best combination for regeneration explained in this study is one of such protocols

Table_1_In silico characterization of differentially expressed short-read nucleotide sequences identified in dieback stress-induced transcriptomic analysis reveals their role as antimicrobial peptides.docx

We investigated the in silico characterization of short-length nucleotide sequences that were differentially expressed in dieback stress-induced transcriptomic analysis. They displayed homology with C-terminal flanking peptides and defensins-like proteins, revealing their antimicrobial activity. Their predicted fingerprints displayed protein signatures related to antimicrobial peptides. These short-length RGAs have been shown to possess structural motifs such as APLT P-type ATPase, casein kinase II (CK2), protein kinase 3, protein kinase C (PKC), and N-glycosylation site that are the attributes of disease resistance genes. The prediction of arginine and lysine residues in active binding sites in ligand docking analysis prophesied them as antimicrobial peptides due to their strong relation with antimicrobial activity. The in silico structural–functional characterization has predicted their role in resistance against microbial pathogens. Moreover, the predicted antimicrobial peptide regions showed their homology with the signature domain of PR-5-like protein and AMP family Thaumatin</p

DataSheet1_Identification of resistance gene analogs of the NBS-LRR family through transcriptome probing and in silico prediction of the expressome of Dalbergia sissoo under dieback disease stress.docx

Dalbergia sissoo is an important timber tree, and dieback disease poses a dire threat to it toward extinction. The genomic record of D. sissoo is not available yet on any database; that is why it is challenging to probe the genetic elements involved in stress resistance. Hence, we attempted to unlock the genetics involved in dieback resistance through probing the NBS-LRR family, linked with mostly disease resistance in plants. We analyzed the transcriptome of D. sissoo under dieback challenge through DOP-rtPCR analysis using degenerate primers from conserved regions of NBS domain-encoded gene sequences. The differentially expressed gene sequences were sequenced and in silico characterized for predicting the expressome that contributes resistance to D. sissoo against dieback. The molecular and bioinformatic analyses predicted the presence of motifs including ATP/GTP-binding site motif A (P-loop NTPase domain), GLPL domain, casein kinase II phosphorylation site, and N-myristoylation site that are the attributes of proteins encoded by disease resistance genes. The physicochemical characteristics of identified resistance gene analogs, subcellular localization, predicted protein fingerprints, in silico functional annotation, and predicted protein structure proved their role in disease and stress resistance.</p