Sangram Sinha

IN VITRO DIFFERENTIATION AND PLANT REGENERATION OF ALBIZIA CHINENSIS (OSB.) MERR.

SummaryPetiolar and distal cotyledonary segments (PCS and DCS) of Albizia chinensis were cultured on Murashige and Skoogs (MS; 1962) medium and induced to form adventitious shoot buds in the presence of either cytokinins 6-benzylamino purine (BAP), kinetin (KN) or thidiazuron (TDZ). Superiority of BAP in inducing shoot bud and differentiation was observed. PCS was more morphogenic to shoot bud differentiation than DCS. TDZ was highly effective in inducing shoot buds, but arrested shoot growth, while KN produced more callus during differentiation of shoots. Rapid and high rate of shoot multiplication per explant was achieved through subculture in MS medium containing BAP (1.0 mg l−1) and indole-3-acetic acid (IAA) (0.5 mg l−1). BAP at low concentration was required to enhance shoot multiplication and elongation. Successful rooting of regenerated shoots was carried out in a two-step culture procedure in MS media with indole-3-butyric acid (IBA) (2.0 mg l−1) and subsequent subculture in IBA-free medium.

Development of a DNA sensor using a molecular logic gate.

This communication reports the increase in fluorescence resonance energy transfer (FRET) efficiency between two laser dyes in the presence of deoxyribonucleic acid (DNA). Two types of molecular logic gates have been designed where DNA acts as input signal and fluorescence intensity of different bands are taken as output signal. Use of these logic gates as a DNA sensor has been demonstrated.

Average packing ratio as a parameter for analyzing the karyotypes of dioecious cucurbits

Abstract Chromosome studies and in situ estimation of 4C nuclear DNA content were carried out on dioecious Coccinia indica and Trichosanthes dioica to understand the differential condensation of mitotic chromosome in relation to sex. The somatic chromosome number for each sex form of C. indica and T. dioica was found to be 2n = 24 and 22 respectively. The karyotypes of the sex forms of both species show high homogeneity, though a distinct hetermorphic pair of sex chromosomes is found in male plants of C. indica. Interrelationship between the 4C nuclear content and the chromosome length has been explained in terms of average packing ratio. The data suggests that average packing ratio is a determinant of distinction between two sexes and therefore could be used as a parameter for analyzing the karyotypes of dioecious plants.

HIGH-FREQUENCY INDUCTION OF MULTIPLE SHOOTS AND CLONAL PROPAGATION FROM RHIZOMATOUS NODAL SEGMENTS OF HOUTTUYNIA CORDATA THUNB. – AN ETHNOMEDICINAL HERB OF INDIA

SummaryThis study reports an efficient and direct shoot bud differentiation and multiple shoot induction from nodal segments of underground stoloniferous rhizomes of Houttuynia cordata Thumb. The frequency of shoot bud regeneration was influenced by the type of cytokinin and concentrations. Among the various concentrations used, benzylaminopurine (BAP, 17.74 μM) or kinetin (Kn, 18.58 μM) was found to be most effective for rapid and maximum shoot but differentiation. The number of shoots per explant was higher (20.00±2.61) on Murashige and Skoog (MS) medium supplemented with Kn (18.58 μM) compared to BAP and 6-γ-γ-(dimethyl-allylamino)-purine (2iP) during initial 40-d-old culture. Subsequent shoot differentiation and multiplication were achieved in MS medium containing 9.29 μM Kn and 15% (v/v) coconut milk. Elongation and growth of multiple shoots were also obtained on MS medium containing either 2.32 μM Kn or 2.46 μM 2iP alone. The rate of shoot multiplication during subcultures declined with an increase in the size of proliferating shoot cluster. Reducing shoot cluster size to three to four shoots and subculturing together in shoot multiplication medium resulted in a better shoot multiplication and growth, which could be maintained for 2 yr. The elongated shoots (>20 mm) were successfully rooted on MS medium supplemented with 19.60 μM indole-3-butyric acid. Regenerated plants were successfully established in soil and were found to be healthy and uniform. The protocol reported in this study can be used for conservation and utilization of elite clone of H. cordata.

Flower development, pollen fertility and sex expression analyses of three sexual phenotypes of Coccinia grandis

BackgroundCoccinia grandis is a dioecious species of Cucurbitaceae having heteromorphic sex chromosomes. The chromosome constitution of male and female plants is 22 + XY and 22 + XX respectively. Y chromosome of male sex is conspicuously large and plays a decisive role in determining maleness. Sex modification has been studied in hypogynous Silene latifolia (Caryophyllaceae) but there is no such report in epigynous Coccinia grandis. Moreover, the role of organ identity genes during sex expression in Coccinia has not been evaluated earlier. Investigations on sexual phenotypes of C. grandis including a rare gynomonoecious (GyM) form and AgNO3 mediated sex modification have added a new dimension to the understanding of sex expression in dioecious flowering plants.ResultsMorphometric analysis showed the presence of staminodes in pistillate flowers and histological study revealed the absence of carpel initials in male flowers. Though GyM plant had XX sex chromosomes, the development of stamens occurred in hermaphrodite flowers but the pollens were not fertile. Silver nitrate (AgNO3) application enhanced stamen growth in wild type female flowers like that of GyM plant but here also the pollens were sterile. Differential expression of CgPI could be involved in the development of different floral phenotypes.ConclusionsThe three principle factors, Gynoecium Suppression (SuF), Stamen Promoting Factor (SPF) and Male Fertility (mF) that control sex expression in dioecious C. grandis assumed to be located on Y chromosome, play a decisive role in determining maleness. However, the characteristic development of stamens in hermaphrodite flowers of GyM plant having XX sex chromosomes indicates that Y-linked SPF regulatory pathway is somehow bypassed. Our experimental findings together with all other previous chromosomal and molecular cytogenetical data strongly support the view that C. grandis could be used as a potential model system to study sex expression in dioecious flowering plant.

De novo transcriptome assembly from flower buds of dioecious, gynomonoecious and chemically masculinized female Coccinia grandis reveals genes associated with sex expression and modification

BackgroundCoccinia grandis (ivy gourd), is a dioecious member of Cucurbitaceae having heteromorphic sex chromosomes. Chromosome constitution of male and female plants of C. grandis is 22A + XY and 22A + XX respectively. Earlier we showed that a unique gynomonoecious form of C. grandis (22A + XX) also exists in nature bearing morphologically hermaphrodite flowers (GyM-H). Additionally, application of silver nitrate (AgNO3) on female plants induces stamen development leading to the formation of morphologically hermaphrodite flowers (Ag-H) despite the absence of Y-chromosome. Due to the unavailability of genome sequence and the slow pace at which sex-linked genes are identified, sex expression and modification in C. grandis are not well understood.ResultsWe have carried out a comprehensive RNA-Seq study from early-staged male, female, GyM-H, and Ag-H as well as middle-staged male and GyM-H flower buds. A de novo transcriptome was assembled using Trinity and annotated by BLAST2GO and Trinotate pipelines. The assembled transcriptome consisted of 467,233 ‘Trinity Transcripts’ clustering into 378,860 ‘Trinity Genes’. Female_Early_vs_Male_Early, Ag_Early_vs_Female_Early, and GyM-H_Middle_vs_Male_Middle comparisons exhibited 35,694, 3574, and 14,954 differentially expressed transcripts respectively. Further, qRT-PCR analysis of selected candidate genes validated digital gene expression profiling results. Interestingly, ethylene response-related genes were found to be upregulated in female buds compared to male buds. Also, we observed that AgNO3 treatment suppressed ethylene responses in Ag-H flowers by downregulation of ethylene-responsive transcription factors leading to stamen development. Further, GO terms related to stamen development were enriched in early-staged male, GyM-H, and Ag-H buds compared to female buds supporting the fact that stamen growth gets arrested in female flowers.ConclusionsSuppression of ethylene responses in both male and Ag-H compared to female buds suggests a probable role of ethylene in stamen suppression similar to monoecious cucurbits such as melon and cucumber. Also, pollen fertility associated GO terms were depleted in middle-staged GyM-H buds compared to male buds indicating the necessity of Y-chromosome for pollen fertility. Overall, this study would enable identification of new sex-biased genes for further investigation of stamen arrest, pollen fertility, and AgNO3-mediated sex modification.

Variation in Leaf Nitrate Reductase Activity during Phenological Stages of Hyptis suaveolens (L.) Poit.: An Ethnomedicinal weed of Tripura, India

Leaf Nitrate Reductase activity was studied in Hyptis suaveolens (L.) Poit. at different phenological stages in relation to leaf position. Variability in nitrate reductase activity at different stages of growth coincident with seasonal changes was recorded. Least amount of nitrate reductase activity was observed in young vegetative stage during summer. This was followed by maximum nitrate reductase activity (4.90 ± 1.47μmole NO2 g−1f.wt.h−1) in mature vegetative stage during rainy season and subsequent decline at flowering stage. Increased activity of nitrate reductase was also observed during post flowering stage of fruit setting in winter. Irrespective of phenological stages, apical leaves exhibited moderately high NRA. High nitrate reductase activity in mature vegetative stage of H. suaveolens characterizes an enhanced potential for nitrate assimilation ascribing to invasive nature of the weed. Present finding highlights a possible impact of morphophysiological status of the plant coinciding with seasonal changes on leaf nitrate reductase activity.

In Vitro Clonal Propagation of Musa Sp. Cultivar Gopi: A Palatable Banana of Tripura, India

The present investigation has been concentrated on the establishment of an efficacious method for in vitro regeneration of a Musa cultivar Gopi of Tripura. Focus has been greatly anchored to the sterilization procedure for assuring successful aseptic culture establishment of this cultivar. The culture protocol for shoot induction and proliferation comprised of four different treatments in basal MS medium fortified with 6-Benzylaminopurine of varying concentrations. The optimum response was encountered in the treatment of BAP in MS medium at concentration of 8mg l -1 for shoot bud induction and multiplication. Subsequent shoot growth and multiplication was achieved through repeated subcultures in media containing lower concentration (4 mg l -1 ). Root initiation was stimulated and brought about by treatments with two different concentrations of Indole butyric acid. The best rooting was manifested in treatment with IBA at concentration of 2mg l -1 . The present study is an approach towards successful establishment of a simple and rapid clonal propagation of a potent Musa cultivar Gopi having traditional and commercial value from this region of Tripura, North-east India.

Karyomorphology of Solanum viarum Dunal-An Ethnomedicinal species of Tripura

The karyomorphological analysis of Solanum viarum Dunal showed the somatic chromosome count to be 2n=24 with n=12 normal bivalents which corroborates previous report. The chromosomes are small with an average size 2.54 ±0.18μm having karyotype formula 2A+4B+12C+6D. The karyotype is symmetric in nature and falls under “1A” of Stebbins symmetric index. The somatic chromosome count and constancy of the karyotype recorded in the present population could be utilised in exploiting cytological and karyomorphological variability among the ecotypic populations of the species.