Brijmohan Singh Bhau

Analysis of Genetic Diversity of Persea bombycina “Som” Using RAPD-Based Molecular Markers

The utility of RAPD markers in assessing genetic diversity and phenetic relationships in Persea bombycina, a major tree species for golden silk (muga) production, was investigated using 48 genotypes from northeast India. Thirteen RAPD primer combinations generated 93 bands. On average, seven RAPD fragments were amplified per reaction. In a UPGMA phenetic dendrogram based on Jaccard’s coefficient, the P. bombycina accessions showed a high level of genetic variation, as indicated by genetic similarity. The grouping in the phenogram was highly consistent, as indicated by high values of cophenetic correlation and high bootstrap values at the key nodes. The accessions were scattered on a plot derived from principal correspondence analysis. The study concluded that the high level of genetic diversity in the P. bombycina accessions may be attributed to the species’ outcrossing nature. This study may be useful in identifying diverse genetic stocks of P. bombycina, which may then be conserved on a priority basis.

Role of Azospirillum in the Improvement of Legumes

A rapid and sustained increase in crop production is one of the essential steps to meet the food demands of the growing human population all over the world. Constant and unbalanced use of chemical fertilizers used to boost the crop productivity on the other hand is causing decrease in nutrient uptake leading to decreased crop yields. Moreover, the use of chemical fertilizers has several other drawbacks, like they are expensive and cause ground water, soil, and atmospheric pollution. Long-term sustainability in agriculture is possible only through the use of low cost farm grown inputs, which work in harmony with the nature. Biofertilizers in this regard act as perpetually responsible input helping in better maintenance of crop nutrient as well as soil health. Azospirillum, an associative symbiotic nitrogen-fixing bacterium, has a higher nitrogen-fixing potential. Though, a number of papers have highlighted the potential application of this microbe, further more research is still needed on Azospirillum to understand the mechanism by which the introduced microorganism benefit the crop. The present review addresses the central issues of Azosprillum application either alone or in combination with other plant growth-promoting rhizobacteria for the benefit of the crops.

Development of an effective and efficient DNA isolation method for Cinnamomum species

Different species of Cinnamomum are rich in polysaccharides and secondary metabolites, which hinder the process of DNA extraction. High quality DNA is the pre-requisite for any molecular biology study. In this paper we report a modified method for high quality and quantity of DNA extraction from both lyophilized and non-lyophilized leaf samples. Protocol reported differs from the CTAB procedure by addition of higher concentration of salt and activated charcoal to remove the polysaccharides and polyphenols. Wide utility of the modified protocol was proved by DNA extraction from different woody species and 4 Cinnamomum species. Therefore, this protocol has also been validated in different species of plants containing high levels of polyphenols and polysaccharides. The extracted DNA showed perfect amplification when subjected to RAPD, restriction digestion and amplification with DNA barcoding primers. The DNA extraction protocol is reproducible and can be applied for any plant molecular biology study.

Microsporogenesis and pollen formation in Zingiber officinale Roscoe

Zingiber officinale Roscoe, the common ginger, is an invaluable horticultural crop cultivated majorly in China, India, Brazil, Jamaica and Nigeria. Its pungent aromatic rhizome is used all over the world as a spice, culinary herb, condiment, home remedy and medicinal agent. This species does not produce seeds and so is difficult to breed its new genotypes through sexual hybridization. Despite the economic and genetic value, ginger has not been subjected to detailed cytogenetic research, which could lead to a better understanding of its reproduction for future genetic improvement. Therefore, the present work is undertaken for the first time to study microsporogenesis and pollen formation in Z. officinale. Pre-meiotic stages were observed under light microscope using anther squashes and staining by either acetocarmine or carbol fuchsin, whereas, the post meiotic stages were visualized under fluorescence microscope using either isolated microspores or pollen grains stained by 4′, 6′-diamidino-2-phenylindole. The combination of these two different cytological techniques allowed the study of the complete microsporogenesis and pollen formation processes step by step starting from pollen mother cells (PMCs) stage to mature pollen grain. Assessment of meiotic behavior was also performed by evaluating all possible PMCs on each slide and at all stages of meiosis. Cytological analysis revealed that only anthers from flowers of small spikes presented appropriate PMCs to analyze meiotic irregularities. The present paper enriches the database of cytology and pollen viability estimations for supporting sexual hybridization. The present study demonstrated a good relationship between the development stage of microspore and the size of the spike. This basic information will hopefully allow the development of protocol for production of androgenic haploids to accelerate breeding and genetic improvement of ginger. Further, research is under progress for evaluation of several media and other cultural factors to achieve microspore embryogenesis and plant regeneration in this species.

Development and characterization of polymorphic microsatellites markers in endangered Aquilaria malaccensis

Aquilaria malaccensis is a critically endangered species in Asia because of its overexploitation to obtain high valued agarwood, used in medicine, perfume, and incense production. Its genetic improvement faces serious problem due to lack of genomic resources. In this study, eighteen novel highly polymorphic microsatellite markers derived from enriched genomic libraries were characterized using 45 individuals of three populations, would be an asset to its non-existing marker recourse. Allelic variations per locus ranged from 2 to 6 with mean allele number was 3.28. The average observed and expected heterozygosity varied from 0.811 and 0.597, respectively. Reproducibility rate, polymorphic potential and ability to detect heterozygosity, novel microsatellites markers would contribute in evaluation of genetic diversity and population structure, and selection of elite germplasm for conservation of genetic resources and commercial utilization.

High gene flow and genetic diversity in three economically important Zanthoxylum Spp. of Upper Brahmaputra Valley Zone of NE India using molecular markers.

The genetic diversity in Zanthoxylum species viz. Zanthoxylum nitidum, Zanthoxylum oxyphyllum and Zanthoxylum rhesta collected from the Upper Brahmaputra Valley Zone of Assam (NE India) was amplified using 13 random amplified polymorphic DNA (RAPD) markers and 9 inter-simple sequence repeat (ISSR) markers. RAPD markers were able to detect 81.82% polymorphism whereas ISSR detected 98.02% polymorphism. The genetic similarities were analyzed from the dendrogram constructed by RAPD and ISSR fingerprinting methods which divided the 3 species of Zanthoxylum into 3 clear different clusters. The principle component analysis (PCA) was carried out to confirm the clustering pattern of RAPD and ISSR analysis. Analysis of molecular variance (AMOVA) revealed the presence of significant variability between different Zanthoxylum species and within the species by both RAPD and ISSR markers. Z. nitidum was found to be sharing a high degree of variation with the other two Zanthoxylum species under study. The Neis gene diversity (h), Shannons information index (I), observed number of alleles (na) and effective number of alleles (ne) were also found to be higher in ISSR markers (0.3526, 0.5230, 1.9802 and 1.6145) than in RAPD markers (0.3144, 0.4610, 1.8182 and 1.5571). The values for total genotype diversity for among population (HT), within population diversity (Hs) and gene flow (Nm) were more in ISSR (0.3491, 0.2644 and 1.5610) than RAPD (0.3128, 0.2264 and 1.3087) but the mean coefficient of gene differentiation (GST) was more in RAPD (0.2764) than ISSR (0.2426). A comparison of this two finger printing methods was done by calculating MR, EMI and MI. The correlation coefficient between data matrices of RAPD and ISSR based on Mantel test was found to be significant (r = 0.65612).

A Novel Tool of Nanotechnology: Nanoparticle Mediated Control of Nematode Infection in Plants

The designing and synthesis of nanoparticle research has been established as an area of intense and dynamic scientific area of research and academia. Due to their unusual physical and chemical properties, nanoparticles have drawn a tremendous amount of attention. Nanobiotechnology holds the promise of controlled release and site-targeted delivery of agrochemicals. A plethora of chemical, physical and biological techniques continues to evolve leading to the production of noble metal nanoparticles. Alongside, biological organisms including plant, fungi and bacteria are an ideal source for green synthesis of nanoparticles with desired shape and size. Some of these nanoparticles also have nematicidal properties, which apply to numerous genera of plant parasitic nematodes and also to plant pathogenic fungi and bacteria. Plant parasitic nematodes are major agricultural pests causing crop losses worth hundreds of billions dollars annually worldwide. Traditional control measures depend upon highly toxic nematicides. In theory, seed treatment for nematode control is optimal, but is largely ineffectual due to poor rhizosphere delivery. Active ingredients of various nanoparticles have also shown evidence of being potentially effective nematicides, which makes these nanoparticles a suitable noble source to control nematode infection in plants. Although very limited reports are available on the use of nanoparticles to control plant nematodes, very encouraging reports are there and research in this area is getting lot of attention. This chapter focuses on the nanoparticles, their synthesis, properties and their use to control nematode infection in plants.

Genetic diversity and population structure of endangered Aquilaria malaccensis revealed potential for future conservation

The endangered Aquilaria malaccensis, is an important plant with high economic values. Characterization of genetic diversity and population structure is receiving tremendous attention for effective conservation of genetic resources. Considering important repositories of biological diversity, the genetic relationships of 127 A. malaccensis accessions from 10 home gardens of three states of northeast India were assessed using amplified fragment length polymorphism (AFLP). Of the 1153 fragments amplified with four AFLP primer combinations, 916 (79.4%) were found to be polymorphic. Polymorphic information content (PIC) and marker index (MI) of each primer combination correlate significantly with the number of genotypes resolved. Overall, a high genetic diversity (avg. 71.85%) was recorded. Further, high gene flow (Nm: 3.37), low genetic differentiation (FST: 0.069) and high within population genetic variation (93%) suggests that most of the genetic diversity is restricted within population. Neighbour joining (NJ), principal coordinate analysis (PCoA) and Bayesian-based STRUCTURE grouped all the accessions in two clusters with significant intermixing between populations, therefore, revealed that two genetically distinct gene pools are operating in the A. malaccensis populations cultivated in home gardens. Based on the various diversity inferences, five diverse populations (JOH, FN, HLF, DHM and ITN) were identified, which can be potentially exploited to develop conservation strategies for A. malaccensis.

DNA barcoding of the genus Nepenthes (Pitcher plant): a preliminary assessment towards its identification

BackgroundDNA barcoding is impending towards the generation of universal standards for species discrimination with a standard gene region that can be sequenced accurately and within short span of time. In this study, we were successful in developing efficient barcode locus in the Nepenthes genus. A total of 317 accessions were retrieved from GenBank of NCBI which represent 140 different species Nepenthes and evaluated the efficacy of ITS, rbcl and matK barcode candidates using barcode gap, applied distance similarity, and tree-based methods.ResultOur result indicates that single-locus ITS or combined with plastid regions (matK) showed the best species discrimination with distinctive barcoding gaps. Therefore, we tentatively proposed the combination of ITS+matK as a core barcode for Nepenthes genus.ConclusionThis study provides a report on DNA barcoding for unique insectivores’ Nepenthes genus. As the different species of Nepenthes are higly endemic and endangered, it would be a useful study to understand the evolutionary relationship, sketched in emigration, mislabeling and can be a probable assessment for its biodiversity.

An Expedition to the Mechanism of Plant–Microbe Interaction by Utilization of Different Molecular Biology Tools

The global demand for food, animal feed, and plant-based products is increasing with the blast of population growth putting unprecedented pressure to the agriculture as the natural resources become diminished and the conventional system of cultivation is not sufficient to cope up with this. In addition to this, recent public concerns to the catastrophic effect of chemical fertilizer and pesticides to the livestocks and the environment led to the urgency of adopting sustainable agricultural practices. In sustainable agriculture, the plant–microbe interaction plays an imperative position which mainly confers the mechanism and utilization of beneficial microbes and their products for crop improvement, providing abiotic stress tolerance and control of plant diseases. The interaction between plants and microbes is a very complex and dynamic biological process which has evolved due to thousand years of coevolution between them. The plant–microbe interactions can provide the new imminent in various aspects of the mechanisms of how the microbes respond to perturbation, how chemical exudates released from plant roots, and how do they affect plant health and development. In the last two decades, molecular biology is being a powerful and precise tool becoming more commonly adopted and reliable for understanding of the plant–microbe interaction. For example, the introduction of next-generation sequencing giving multitude of nucleotide data in a very short duration also assists metagenomics which allows studying complete microbiota including non-culturable microbes. This book chapter is intended to chronicle the development of different molecular biology tools in studying the biosynthetic pathway secondary metabolites produced by microbes, diversity of microorganisms, and functional identification of induced genes in a plant–microbe interaction.