MIR159 regulates multiple aspects of stamen and carpel development and requires dissection and delimitation of differential downstream regulatory network for manipulating fertility traits.

Unravelling genetic networks regulating developmental programs are key to devising and implementing genomics assisted trait modification strategies. It is crucial to understand the role of small RNAs, and the basis of their ability to modify traits. MIR159 has been previously reported to cause defects in anther development in Arabidopsis; however, the complete spectrum and basis of the defects remained unclear. The present study was therefore undertaken to comprehensively investigate the role of miR159 from Brassica juncea in modulating vegetative and reproductive traits. Owing to the polyploid nature of Brassica, paralogous and homeologous copies of MIR159A, MIR159B, and, MIR159C were identified and analysis of the precursor uncovered extensive structural and sequence variation. The MIR159 locus with mature miR159 with perfect target complimentarily with MYB65, was cloned from Brassica juncea var. Varuna for functional characterization by generating constitutively over-expressing lines in Arabidopsis thaliana Col-0. Apart from statistically significant difference in multiple vegetative traits, drastic differences were observed in stamen and pistil. Over-expression of miR159a led to shortening of filament length and loss of tetradynamous condition. Anthers were apiculate, with improper lobe formation, and unsynchronized cellular growth between connective tissue and another lobe development. Analysis revealed arrested meiosis/cytokinesis in microspores, and altered lignin deposition pattern in endothecial walls thus affecting anther dehiscence. In the gynoecium, flaccid, dry stigmatic papillae, and large embryo sac in the female gametophyte was observed. Over-expression of miR159a thus severely affected pollination and seed-set. Analysis of the transcriptome data revealed components of regulatory networks of anther and carpel developmental pathway, and lignin metabolism that are affected. Expression analysis allowed us to position the miR159a-MYB65 module in the genetic network of stamen development, involved in pollen-grain maturation; in GA-mediated regulation of stamen development, and in lignin metabolism. The study, on one hand indicates role of miR159a-MYB65 in regulating multiple aspects of reproductive organ development that can be manipulated for trait modification, but also raises several unaddressed questions such as relationship between miR159a and male-meiosis, miR159a and filament elongation for future investigations. Accession numbers: KC204951-KC204960. Project number PRJNA1035268. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01377-7.

Micropropagation of Ajuga bracteosa, a medicinal herb.

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25 ± 2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.

Evaluation of the catalase promoter for expressing the alkaline xylanase gene (alx) in Aspergillus niger.

Aspergillus niger represents a promising host for the expression of recombinant proteins, but only a few expression systems are available for this organism. In this study, the inducible catalase promoter (PcatR) from A. niger was characterized. For this, constructs were developed and checked for the expression of the alkaline xylanase gene transcriptionally fused under the cat R promoter. Two versions of the catalase (catR) promoter sequence from A. niger (P(cat300,) P(cat924)) were isolated and tested for their ability to drive expression of the alkaline xylanase (alx) gene. P(cat924) showed better efficiency (more than 10-fold increase in AlX activity compared to P(cat300)) under the optimized culture conditions. Induction of the catR promoter with 0.20% H(2)O(2) and 1.5% CaCO(3) in the culture medium, further increased expression of AlX 2.61- and 2.20-fold, respectively, clarifying its inducible nature. Specific induction or repression of the catR promoter provides the possibility for utilization of this promoter in heterologous protein production.

Sesamol as a potential radioprotective agent: in vitro studies.

Protection against radiation-induced DNA strand breaks is an important aspect in the design and development of a radioprotector. In this study, the radioprotective efficacy of sesamol, a natural antioxidant, was investigated in aqueous solution of plasmid DNA (pBR322) and compared with that of melatonin, a known antioxidant-based radioprotector. Thermal denaturation studies on irradiated calf thymus DNA were also carried out with sesamol and melatonin. Sesamol demonstrated greater radioprotective efficacy in both plasmid DNA and calf thymus DNA. To assess the radical scavenging capacity of sesamol and melatonin, 2-deoxyribose degradation, DPPH and ABTS assays were performed. Sesamol exhibited more scavenging capacity compared to melatonin. In vitro studies with V79 cells showed that sesamol is 20 times more potent than melatonin. It is proposed that the greater radioprotective efficacy of sesamol could be due to its greater capacity for scavenging of free radicals compared to melatonin. The results will be helpful in understanding the mechanisms and development of sesamol as a radioprotector.

Biological and Molecular Characterization of Two Distinct Tomato Strains of Cucumber mosaic virus Based on Complete RNA-3 Genome and Subgroup Specific Diagnosis.

Cucumber mosaic virus (CMV) is one of the most important viral pathogen infecting several plant species in India. Five isolates of CMV obtained from cucumber, muskmelon, tobacco and tomato from distinct geographical locations in India were analysed based on host-reactions and genome sequence. The majority of the isolates were very similar and only two isolates, Tfr-In and Tss-In showed distinct symptoms in tomato and high sequence diversity (77.8%) in coat protein (CP) gene. Tfr-In was isolated from tomato fruit showing grey patches in Aurangabad and Tss-In from tomato plant showing shoe-string symptoms in New Delhi. The RNA-3 genomes of Tfr-In (2,214 nt; JF279606), shared only 70.3% nucleotide sequence identity with Tss-In (2,178 nt; JF279605. The complete RNA-3 genome of Tss-In and Tfr-In were compared with that of 65 CMV isolates reported from various plants of the world, which formed four distinct subclades-IA, -IB, -IC and -II. The Tfr-In isolate clustered with the CMV subgroup-IB and Tss-In with the subgroup-II. The comparison of the RNA-3 sequence of both the isolates revealed maximum heterogeneity in the intergenic region (IR). Reverse transcriptase polymerase chain reaction (RT-PCR) based detection of CMV subgroup-I and -II was developed designing primers from flanking IR region. The specificity of the RT-PCR detection was confirmed using Tfr-In and Tss-In representing subgroup-I and -II and validated with field samples of tomato, cucurbits and chilli. This is the first report of complete RNA-3 of subgroup-IB CMV causing grey patches in tomato fruit and subgroup-II CMV causing shoe-string symptoms in tomato in India. The present and previous studies together showed that tomato in India was affected by multiple strains of CMV.

Transgenic tobacco plant expressing environmental E. coli merA gene for enhanced volatilization of ionic mercury.

The practicability of transgenic tobacco engineered to express bacterial native mercuric reductase (MerA), responsible for the transport of Hg2+ ions into cell and their reduction to elemental mercury (Hg0), without any codon modification for phytoremediation of mercury pollution was evaluated. Transgenic tobacco plants reduce mercury ions to the metallic form; take up metallic mercury through their roots; and evolve less toxic elemental mercury. Transformed tobacco produced a large amount of merA protein in leaves and showed a relatively high resistant phenotype to HgCl2 than wild-type. Results suggests that the integrated merA gene, encoding mercuric reductase, a key enzyme of bacterial mer operon, is stably integrated into tobacco genome, and translated to active MerA which catalyzed the bioconversion of toxic Hg2+ to least toxic elemental Hg0, and suggest that MerA is capable of reducing the Hg2+, probably via NADPH as an electron donor. The transgenic tobacco expressing merA volatilized significantly more mercury than wild-type plants. This is first time we are reporting the expression of bacterial native merA gene via the nuclear genome of Nicotiana tabacum and enhanced mercury volatilization from tobacco transgenics. The study clearly indicates that transgenic tobacco plants are reasonable candidates for the remediation of mercury contaminated areas.

Difference in in vitro response and esculin content in two populations of Taraxacum officinale Weber.

In vitro micropropagation has been achieved in medicinally important plant, Taraxacum officinale collected from two different regions, Kashmir (J & K) and Garhwal (Uttarakhand). Leaf segments inoculated on MS supplemented with different combinations of Indole-3-acetic acid (IAA) and Benzyladenine (BA) produced indirect regeneration. For root induction MS fortified with Indole-3-butyric acid (IBA) was used. Taraxacum officinale collected from Garhwal responded two weeks earlier and showed shoot regeneration whereas in Kashmir population only callus proliferation occurred. Esculin content was also higher in the samples from Garhwal. The content was affected by both, the hormone concentration as well as age of the cultures. RAPD of the in vitro raised regenerants confirmed genetic stability.

A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens.

Proliferation of axillary shoot buds and multiple shoot formation in Catharanthus roseus was obtained in 96 % explants on MS medium (3 % sucrose) containing NAA + BA. 2,4-D induced callusing in both, the nodal as well as in leaf segments. Leaf-derived callus was used for transformation with Agrobacterium tumefaciens LBA4404/pBI-S1. Bacterial cell concentration, duration of co-cultivation and acetosyringone concentration influenced transformation efficiency. Under optimal co-cultivation conditions, 98 % of the explants showed GUS expression. PCR based amplification of the transformed and subsequently selected callus tissue indicated the presence of uidA, Gly I and nptII genes.

Cryopreservation of embryogenic cultures of Dioscorea bulbifera l. by encapsulation- dehydration.

Embryogenic cultures of Dioscorea bulbifera were cryopreserved using an encapsulation- dehydration procedure with subsequent plant regeneration. Embryogenesis was induced by culturing in vitro grown axillary bud meristems on MS medium supplemented with 2.0 mg per liter 2,4-D. After cryopreservation, recovery growth of embryogenic culture up to 53.3 percent was recorded when excised proliferating embryogenic cultures of 1.5-2.0 mm in diameter were: encapsulated in 3 percent calcium alginate containing 0.15 M sucrose followed by preculturing with 0.5 M sucrose for 3 d; dehydrated in the laminar air flow for 4 h, thereby reducing the bead moisture content to 19.4 percent ( fresh weight basis); plunged into liquid nitrogen; thawed at 40 degree C; and cultured on recovery growth medium, i.e. MS supplemented with 2.0 mg per liter 2,4-D and 0.3 mg per liter BAP. However, preculturing for an extended period of 7 d increased the recovery growth further to 67.8 percent. During recovery growth the embryogenic tissue protruded out of the beads without loss of structural integrity of the cryopreserved embryos. Subculturing of these cultures on to embryo conversion medium, i.e. MS medium with 0.5 mg per liter zeatin and 400 mg per liter glutamine, resulted in production of plantlets through embryo conversion. The regenerated plantlets exhibited the same morphology as that of originally maintained in vitro plantlets and were established in vivo, in a net house with 80 percent success.

Cryopreservation of Dioscorea rotundata poir.: a comparative study with two cryogenic procedures and assessment of true-to-type of regenerants by RAPD analysis.

The aim of this study was to develop a cryopreservation protocol for Dioscorea rotundata with maintenance of genetic stability of regenerated plants after cryopreservation. In vitro shoot tips were cryopreserved using vitrification and encapsulation-dehydration to compare the efficacy of the two methods. Both methods produced high levels of plant regeneration from cryopreserved shoot tips. The regeneration level obtained using vitrification (71%) was not significantly different from that obtained using encapsulation-dehydration (67%). Genetic stability of plants derived from cryopreserved shoot tips was evaluated using RAPD markers. Analysis of 50 cryopreserved-derived and 20 in vitro- maintained (control) plantlets showed that 10 primers produced 77 clear, reproducible bands, with the amplification products being monomorphic for all the plantlets tested. A total of 5,390 bands obtained from this study exhibited no aberration in RAPD banding. Thus, the present study showed that both vitrification and encapsulation-dehydration methods are equally applicable to D. rotundata for cryopreservation. The in vitro plantlets derived from cryopreservation were genetically stable at the molecular level tested.

Analysis of genetic diversity through AFLP, SAMPL, ISSR and RAPD markers in Tribulus terrestris, a medicinal herb.

Tribulus terrestris is well known for its medicinal importance in curing urino-genital disorders. Amplified fragment length polymorphism (AFLP), selective amplification of microsatellite polymorphic loci (SAMPL), inter-simple sequence repeat (ISSR) and randomly amplified polymorphic DNA (RAPD) markers were used for the first time for the detection of genetic polymorphism in this medicinal herb from samples collected from various geographical regions of India. Six assays each of AFLP and SAMPL markers and 21 each of ISSR and RAPD markers were utilized. AFLP yielded 500 scorable amplified products, of which 82.9% were polymorphic. SAMPL primers amplified 488 bands, 462 being polymorphic (94.7%). The range of amplified bands was 66 [(TC)(8)G + M-CAG] to 98 [(CA)(6)AG + M-CAC] and the percentage polymorphism, 89.9 [from (CT)(4)C (AC)(4)A + M-CTG] to 100 [from (GACA)(4) + M-CTA]. The ISSR primers amplified 239 bands of 0.4-2.5 kb, 73.6% showed polymorphism. The amplified products ranged from 5 to 16 and the percentage polymorphism 40-100. RAPD assays produced 276 bands, of which 163 were polymorphic (59%). Mantel test employed for detection of goodness of fit established cophenetic correlation values above 0.9 for all the four marker systems. The dendrograms and PCA plots derived from the binary data matrices of the four marker systems are highly concordant. High bootstrap values were obtained at major nodes of phenograms through WINBOOT software. The relative efficiency of the four molecular marker systems calculated on the basis of multiplex ratio, marker index and average heterozygosity revealed SAMPL to be the best. Distinct DNA fingerprinting profile, unique to every geographical region could be obtained with all the four molecular marker systems. Clustering can be a good indicator for clear separation of genotypes from different regions in well-defined groups that are supported by high bootstrap values.

Genetic fidelity of in vitro regenerants, encapsulation of shoot tips and high diosgenin content in Dioscorea bulbifera L., a potential alternative source of diosgenin.

Dioscorea bulbifera L. containing the pharmaceutically important compound, diosgenin, was regenerated in vitro through nodal segments on supplemented Murashige and Skoog medium (MS). Diosgenin was at 12 mg g(-1)dry wt in 12-week-old plantlets raised on MS with various growth hormones. Random amplified polymorphic DNA (RAPD) analysis showed genetic fidelity of regenerants. Encapsulation of shoot tips in 3% (w/v) calcium alginate for storage and germplasm exchange was achieved.

Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.).

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.

Abiotic metal stress enhances diosgenin yield in Dioscorea bulbifera L. cultures.

Lower concentrations of CuSO(4) (25-75 microM) in the MS medium supplemented with 0.1 mg l(-1) IAA+5.0 mg l(-1) Kn+500 mg l(-1) CH+10 mg l(-1) Cyst hyd enhanced the growth of regenerants of Dioscorea bulbifera L. CuSO(4) (75 microM) induced an appreciable diosgenin yield in the regenerants compared to those obtained on media without Cu. The presence of Cu thus seems to stimulate diosgenin production. The regenerants also differentiated bulbils on lower concentrations of Cu. At CuSO(4) (100 microM), however, cultures showed poor growth as well as a low diosgenin yield. Increased proline and protein contents were recorded in cultures grown on Cu-enriched media.

In vitro micropropagation, differentiation of aerial bulbils and tubers and diosgenin content in Dioscorea bulbifera.

Dioscorea bulbifera could be micropropagated through nodal segments and bulbils. The best medium for regeneration and bulbil differentiation was MS + 0.5 microM IAA (indole-3-acetic acid) + 20.0 microM Kn (kinetin) + 500 mg/L CH (casein hydrolysate) + activated charcoal (20 %). Diosgenin content was maximum in regenerants grown on MS + 5.0 microM IAA + 20.0 microM Kn + 500 mg/L CH. T.s of bulbils could also be used for direct plantlet differentiation as well as bulbil differentiation on MS + 10.0 microM IAA + 20.0 microM Kn + (in mg/L) 30 each of Asp (asparagine) + Arg (arginine) + Gln (glutamine) + 10 Ad (adenine) + 500 CH + 10 Cyst hyd (cysteine hydrochloride). Diosgenin yield in plantlets reached a maximum after 20 weeks. The results indicate that micropropagation, bulbil formation and tuberisation can be achieved in vitro in D. bulbifera, hitherto a less exploited plant, and can further be used for obtaining enhanced levels of diosgenin.

Genetic stability assessment of plants regenerated from cryopreserved embryogenic tissues of Dioscorea bulbifera l. Using RAPD, biochemical and morphological analysis.

Embryogenic tissues of Dioscorea bulbifera were cryopreserved using the encapsulation-dehydration technique. Genetic stability of plants regenerated from cryopreserved embryogenic tissues was assessed using molecular, biochemical and morphological analysis. The random amplified polymorphic DNA (RAPD) analysis of 60 cryopreserved-derived and 20 in vitro grown (control) plantlets showed that 10 primers produced 62 clear reproducible DNA fragment profiles. The amplification products were monomorphic for all the plantlets except one. A total of 4960 DNA fragments were obtained from this study showing no variation in RAPD profiles. The diosgenin content of cryopreserved-derived plants, analyzed using HPLC, was similar to that of control plants. Morphology and the ability to form microtuber were also found to be unaltered in cryopreserved embryo-derived plantlets. Thus, the D. bulbifera plants regenerated from cryopreserved embryogenic tissues were genetically stable at the molecular, biochemical and morphological levels.

Molecular characterization and distribution of a 145-bp tandem repeat family in the genus Populus.

This report aims to describe the identification and molecular characterization of a 145-bp tandem repeat family that accounts for nearly 1.5% of the Populus genome. Three members of this repeat family were cloned and sequenced from Populus deltoides and P. ciliata. The dimers of the repeat were sequenced in order to confirm the head-to-tail organization of the repeat. Hybridization-based analysis using the 145-bp tandem repeat as a probe on genomic DNA gave rise to ladder patterns which were identified to be a result of methylation and (or) sequence heterogeneity. Analysis of the methylation pattern of the repeat family using methylation-sensitive isoschizomers revealed variable methylation of the C residues and lack of methylation of the A residues. Sequence comparisons between the monomers revealed a high degree of sequence divergence that ranged between 6% and 11% in P. deltoides and between 4.2% and 8.3% in P. ciliata. This indicated the presence of sub-families within the 145-bp tandem family of repeats. Divergence was mainly due to the accumulation of point mutations and was concentrated in the central region of the repeat. The 145-bp tandem repeat family did not show significant homology to known tandem repeats from plants. A short stretch of 36 bp was found to show homology of 66.7% to a centromeric repeat from Chironomus plumosus. Dot-blot analysis and Southern hybridization data revealed the presence of the repeat family in 13 of the 14 Populus species examined. The absence of the 145-bp repeat from P. euphratica suggested that this species is relatively distant from other members of the genus, which correlates with taxonomic classifications. The widespread occurrence of the tandem family in the genus indicated that this family may be of ancient origin.

Enzymatic synthesis of artemisinin from natural and synthetic precursors.

To investigate the biosynthetic pathway of artemisinin, an assay system for the determination of activity of the enzymes involved in its synthesis has been developed. Results from these experiments have shown that HEPES provides a better buffer system than Tris-HCl. The enzyme(s) requires Mg2+ and/or Mn2+, and the addition of ATP and NADPH+H+ significantly enhances the enzyme activity. A new substrate, dihydroarteannuin B, has been synthesized that can easily be radiolabeled with high specific activity. It is utilized by the enzyme system and is converted to artemisinin with the same efficiency as the natural substrates. This can be conveniently used as a precursor for elucidation of the pathway for artemisinin biosynthesis.