Integrated metabolomics and transcriptomics analysis highlight key pathways involved in the somatic embryogenesis of Darjeeling tea.

BACKGROUND: Darjeeling tea is a globally renowned beverage, which faces numerous obstacles in sexual reproduction, such as self-incompatibility, poor seed germination, and viability, as well as issues with vegetative propagation. Somatic embryogenesis (SE) is a valuable method for rapid clonal propagation of Darjeeling tea. However, the metabolic regulatory mechanisms underlying SE in Darjeeling tea remain largely unknown. To address this, we conducted an integrated metabolomics and transcriptomics analysis of embryogenic callus (EC), globular embryo (GE), and heart-shaped embryo (HE). RESULTS: The integrated analyses showed that various genes and metabolites involved in the phenylpropanoid pathway, auxin biosynthesis pathway, gibberellin, brassinosteroid and amino acids biosynthesis pathways were differentially enriched in EC, GE, and HE. Our results revealed that despite highly up-regulated auxin biosynthesis genes YUC1, TAR1 and AAO1 in EC, endogenous indole-3-acetic acid (IAA) was significantly lower in EC than GE and HE. However, bioactive Gibberellin A4 displayed higher accumulation in EC. We also found higher BABY BOOM (BBM) and Leafy cotyledon1 (LEC1) gene expression in GE along with high accumulation of castasterone, a brassinosteroid. Total flavonoids and phenolics levels were elevated in GE and HE compared to EC, especially the phenolic compound chlorogenic acid was highly accumulated in GE. CONCLUSIONS: Integrated metabolome and transcriptome analysis revealed enriched metabolic pathways, including auxin biosynthesis and signal transduction, brassinosteroid, gibberellin, phenylpropanoid biosynthesis, amino acids metabolism, and transcription factors (TFs) during SE in Darjeeling tea. Notably, EC displayed lower endogenous IAA levels, conducive to maintaining differentiation, while higher IAA concentration in GE and HE was crucial for preserving embryo identity. Additionally, a negative correlation between bioactive gibberellin A4 (GA4) and IAA was observed, impacting callus growth in EC. The high accumulation of chlorogenic acid, a phenolic compound, might contribute to the low success rate in GE and HE formation in Darjeeling tea. TFs such as BBM1, LEC1, FUS3, LEA, WOX3, and WOX11 appeared to regulate gene expression, influencing SE in Darjeeling tea.

Validation of determinate (dt) gene-based DNA marker in inter-specific hybrid sesame and in-silico analysis of the predicted dt protein structures.

Determinacy is a desirable trait in sesame, an important oilseed crop. We have developed an inter-specific hybrid between basally branched indeterminate cultivated Sesamum indicum genotype and wild S. prostratum with no branching yet synchronous pods on the shoot. The hybrid and a few exotic sesame germplasms were successfully screened with a determinacy (dt) gene-based DNA marker. In-silico translation of the partial coding sequences of the dt gene from the two contrasting parent genotypes revealed an SNP (V159A) in S. prostratum. The predicted cytoplasmic dt protein showed a high resemblance with flowering protein centroradialis. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-022-01135-1.

RNAi-mediated down-regulation of ITPK-2 enhanced inorganic phosphorus and minerals in the transgenic rice.

Phytic acid or Myo-inositol hexakisphosphate is an essential compound for the rice plants. It remains in the form of phytate, a mixed salt of different mineral cations, in the seeds. The phytate breaks down during germination and provides the inorganic phosphorus and mineral ions to the seedlings. However, humans do not get the benefit of those essential ions from rice consumption due to the absence of phytase in the gut. We envisaged down-regulating ITPK, the gene behind the phytic acid biosynthesis so that its low amount would facilitate a greater amount of free mineral ions in the endosperm. Since there are six homologues of rice ITPK, we studied their expression in seeds. Additionally, we undertook an in-silico analysis of the homologous proteins. Considering the results, we selected ITPK-2 for its RNAi-mediated embryo-specific down-regulation to obtain the low phytate rice. We obtained a 37% reduction of phytic acid content accompanied by a nearly three-fold enhancement of inorganic phosphorus in the transgenic seeds. Additionally, the iron and zinc content increased in polished rice grains compared to the wild type. The results also showed that reduced phytic acid content did not affect the germination potential and seedling growth of the transgenic rice.

Transcriptomic dataset of cultivated (Sesamum indicum), wild (S. mulayanum), and interspecific hybrid sesame in response to induced Macrophomina phaseolina infection.

We report here the data of transcriptome sequencing of control and infected sesame genotypes. Sesame is an emerging oilseed crop [1]. The destructive soil-borne fungi Macrophomina phaseolina Tassi (Goid) causes charcoal rot of sesame, leading to high (>50%) yield loss. Most of the high-yielding sesame cultivars (Sesamum indicum) of India are susceptible to charcoal rot. Wild sesame, Sesamum mulayanum shows a high degree of tolerance against many pathogens [2]. We have earlier developed an interspecific hybrid between Indian cultivated sesame and S. mulayanum. The parents and the F6 recombinant constitute the three experimental genotypes in the present report. The seedlings were infected with M. phaseolina. The data of the infected and control (mock-inoculated) transcriptome is presented. The RNA-seq by Illumina NovaSeq 6000 technology generated 2.9 × 108 paired-end reads. We deposited the data in NCBI sequence read archive (SRA) with accession number PRJNA642699. The de novo assembly of clean reads generated 106,295 unigenes with an average length of 1,342 bp covering 1.42 × 108 nucleotides. The screening of 106,295 unigenes with MISA and SAMtools software resulted in the identification of 26,880 simple sequence repeats (SSRs), 90,181 single nucleotide polymorphisms (SNPs), and 25,063 insertion deletions (InDels). Apart from mono-base repeats, di-nucleotides repeats (42.51%) were found to be the most abundant, followed by tri-nucleotides (14.28%) among the SSRs. Subsequently, we have designed 22,494 pairs of primers based on perfect di and tri-nucleotide SSRs. Transitions (Ts, 60%) were the most abundant substitution type among the SNPs followed by transversions type (Tv, 40%), with a Ts/Tv ratio of 1.48. The development of genic-SSR markers and SNP information will pave the way for molecular marker-assisted breeding of sesame for tolerance against charcoal rot.

Allele specific CAPS marker development and characterization of chalcone synthase gene in Indian mulberry (Morus spp., family Moraceae).

Chalcone synthase (CHS) is an essential enzyme in the phenylpropanoid pathway that catalyzes the first step in flavonoid biosynthesis in plants under diverse environmental stress. We have used CHS as a candidate gene in mulberry and developed Single Nucleotide Polymorphism (SNP) based co-dominant Cleaved Amplified Polymorphic Sequence (CAPS) marker associated with the CHS locus. The segregation pattern of the marker was studied in an F1 population derived from a hybridization program between two mulberry genotypes showing polymorphism for the CHS locus. Differential CHS activity of the recombinants has been correlated with the segregation pattern of the marker. Homology modelling and docking studies are performed for both the identified CHS alleles and correlated with respective CHS activity. Phenotyping of Powdery Mildew infected F1 population indicated a probable association with the CAPS marker.

Pineapple [Ananas comosus (L.) Merr].

The efficacy of Agrobacterium-mediated pineapple transformation technique has been improved (mean percentage of transgenic micro-shoots regenerated from initial callus explants up to 20.6%) using a novel encapsulation-based, antibiotic selection procedure. The detailed protocol using a standard plant transformation vector (pCAMBIA1304) as reported in an 'elite' Indian variety (Queen) of pineapple [Ananas comosus (L.) Merr] can be applied to other varieties of pineapple for introgression of target genes.