Sinchan Adhikari

RAPD and ISSR based evaluation of genetic stability of micropropagated plantlets of Morus alba L. variety S-1

Plant regeneration through rapid in vitro clonal propagation of nodal explants of Morus alba L. variety S-1 was established along with genetic stability analysis of regenerates. Axillary shoot bud proliferation was achieved on Murashige and Skoog (MS) medium in various culture regimes. Highest number of shoots (5.62 ± 0.01), with average length 4.19 ± 0.01 cm, was initially achieved with medium containing 0.5 mg/l N6-benzyladenine (BA) and 3% sucrose. Repeated subculturing of newly formed nodal parts after each harvest up to sixth passage, yielded highest number of shoots (about 32.27) per explants was obtained after fourth passage. Rooting of shoots occurred on 1/2 MS medium supplemented with 1.0 mg/1 Indole-3-butyric acid (IBA). About 90% (89.16) of the plantlets transferred to the mixture of sand:soil:organic manure (2:2:1) in small plastic pots acclimatized successfully. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD (random amplified polymorphic DNA) and ISSR (inter-simple sequence repeat). This protocol can be used for commercial propagation and for future genetic improvement studies.

Identification and Validation of a New Male Sex-Specific ISSR Marker in Pointed Gourd (Trichosanthes dioica Roxb.)

The aim of the present study was to develop a genetic sex marker for the pointed gourd (Trichosanthes dioica Roxb.) to allow gender determination at any stage in the life cycle. Screening of genomic DNA with intersimple sequence repeat (ISSR) primers was used to discover sex-specific touch-down polymerase chain reaction (Td-PCR) amplification products. Using pooled DNA from male and female genotypes and 42 ISSR primers, a putative male specific marker (~550 bp) was identified. DNA marker specific to male is an indication of existence of nonepigenetic factors involved in gender development in pointed gourd. The ISSR technique has proved to be a reliable technique in gender determination of pointed gourd genotypes at the seedling phenophase. The sex marker developed here could also be used as a starting material towards sequence characterization of sex linked genes for better understanding the developmental as well as evolutionary pathways in sexual dimorphism.

Hormonal control of sex expression of cucumber (Cucumis sativus L.) with the identification of sex linked molecular marker

Sex differentiation in plants is closely related to the process of plant growth and development. The mechanism that regulates the transition in flowering plants from the vegetative state to reproductive development is poorly understood because the flowering physiology is difficult to study using traditional methods. In the present study, tissue culture system was used as a potential tool to study the sex determination of cucumber to reduce the influence of environmental factors. Major role of gibberellic acid and cytokinins in the regulation of sex expression of cucumber were demonstrated. Gibberellic acid was associated with male sex expression whereas cytokinins found to be associated with the female sex expression. However 6-Benzyl adenine was found to induce hermaphrodite flowers. In the present study, in vitro flowering with different sex expression as well as sex conversion correlates the established fact that minor changes in the balance between phytohormones in the plants with the same genotypic conditions can result in the development of one sex or another. To understand the molecular background of sex expression in cucumber, random amplified polymorphic DNA (RAPD) marker technique was adopted. The result shows unique banding pattern for genome fingerprinting at an early stage of analysis. Two sex linked markers namely OPS250-11 and OPS600-11 were scored for male and bisexual individuals respectively, that are helpful to understand the developmental biology and stable sex expression of cucumber.

Regeneration of plantlets through somatic embryogenesis from root derived calli of Hibiscus sabdariffa L. (Roselle) and assessment of genetic stability by flow cytometry and ISSR analysis

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver–Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants.

Profiling of BABA-induced differentially expressed genes of Zea mays using suppression subtractive hybridization

β-Aminobutyric acid (BABA), a potent chemical priming agent, induces plant resistance to a broad spectrum of biotic and abiotic stresses. Nevertheless, the molecular mechanisms underlying this phenomenon remain poorly understood. Therefore, this study aims to identify rare and differentially expressed transcripts in BABA-primed maize (Zea mays L.) using a suppression subtractive hybridization strategy. Forward subtraction was performed using the cDNA prepared from a BABA primed leaf as a tester and cDNA from the control leaf as the driver. For reverse subtraction, tester cDNA was synthesized from control leaf and driver cDNA from the BABA primed leaf. A total of 626 clones were picked randomly. In total 192 and 204 high quality expressed sequence tags (ESTs) were generated from forward (ZmSF) and reverse SSH library (ZmSR) respectively. Contigs analyses revealed 27 unigenes from the forward subtracted library and 4 unigenes from the reverse subtracted library. The ESTs encoding alpha amylase, inositol monophosphatase 3, tocopherol cyclase, thioredoxin H-type, aspartic proteinase, NADP-malic enzyme and PEP carboxykinase were expressed predominantly in BABA-primed leaf and found to be predominantly associated with major metabolic and biosynthesis events along with seed germination, developmental processes and adaptive responses to environmental stimuli such as drought, salinity, osmotic stress and pathogen attack. In this study, we present the first report of transcriptome analysis of maize leaf exposed to BABA. The expression profiles of candidate genes from subtracted libraries were checked using semi quantitative RT-PCR and further validated by qRT-PCR. Expression profiles largely corroborated the quality and functional diversity of both libraries. In ZmSF, 130 ESTs (67.70%) did not show any significant similarity to any protein in the NCBI database, indicating a significant fraction of novel and rare expressed transcripts that were rapidly and strongly induced in response to BABA. Further research needs to be done for these novel transcripts to elucidate their function in a BABA-potentiated stress adaptation mechanism. Overall, our study adds new insights into the BABA potentiated defense mechanisms in plants, and strengthens the available transcriptome database of maize.

Application of molecular markers in plant genome analysis: a review

Advancement in the field of molecular biology has led to the development of various molecular markers which has revolutionized our understanding of the organization and evolution of plant genomes. Detection of genetic variation in plants offers an opportunity to understand the molecular basis of several biological phenomena. The reliability and efficiency of restriction digestion and polymerase chain reaction based random DNA markers have already proved their utility in taxonomical, evolutionary and ecological studies of plants. Progresses in the field of genomics and transcriptomics have enabled plant researchers to develop molecular markers derived from exon region of the genome which are termed as genic molecular markers (GMMs). GMMs are the part of the cDNA/EST sequences that mainly characterize the functional part of the genome. Next-generation DNA sequencing has also significantly contributed towards development of microRNA specific novel functional markers at the DNA level. This review focuses on the technical aspects of different molecular markers and their applications in the genome analysis.