Gyana Ranjan Rout

Identification of Tinospora cordifolia (Willd.) Miers ex Hook F. & Thomas Using RAPD Markers

Abstract Identified germplasm is an important component for efficient and effective management of plant genetic resources. Traditionally, plant identification has relied on morphological characters like growth habit, floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and genetic variation within 15 clones of Tinospora cordifolia through random amplified polymorphic DNA (RAPD) markers. Analysis was made using forty decamer primers. Out of them, 15 primers were selected and used for identification and genetic relationships within 15 clones. A total of 138 distinct DNA fragments ranging from 0.2 to 3.2 kb were amplified using 15 selected random primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The genetic distance was very close within the clones. Thus, these RAPD markers have the potential for identification of species and characterization of genetic variation within the population. This study will be helpful to know the genetic background of the medicinal plants with high commercial value, and also provides a major input into conservation biology

Micropropagation and assessment of genetic stability in Celastrus paniculatus: An endangered medicinal plant

A highly efficient protocol for in vitro regeneration of an indigenous, endangered medicinal plant Celastrus paniculatus was achieved using nodal explants. Murashige and Skoog (MS) basal medium supplemented with 0.5 mg/L 6-benzylaminopurine (BAP) and 0.1 mg/L naphthaleneacetic acid (NAA) showed maximum percentage of shoot multiplication (83.4%) with 8.2 shoots/explants. Maximum rooting of 73.3% with 4.8 roots/shoot was achieved on half-strength MS media supplemented with 0.5 mg/L indole-3-acetic acid (IAA) and the percentage of survival was 91% after acclimatization. Random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) marker study confirmed genetic stability for in vitro raised explants by showing 100% monomorphism. High multiplication rate associated with genetic stability ensure the efficacy of the present in vitro clonal propagation protocol of this important medicinal plant species.

Identification and genetic variation among Hibiscus species (Malvaceae) using RAPD markers

Abstract Germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or cultivars identification has relied on morphological characters like growth habit or floral morphology like flower colour and other characteristics of the plant. Studies were undertaken for identification and determination of genetic variation within the two species of Hibiscus and 16 varieties of Hibiscus rosa-sinensis L. through random amplified polymorphic (RAPD) markers. Primer screening was made by using the DNA of variety “Prolific”. Genetic analysis was made by using ten selected decamer primers. A total of 79 distinct DNA fragments ranging from 0.3 to 2.5 kb were amplified by using ten selected random decamer primers. The genetic similarity was evaluated on the basis of presence or absence of bands. The cluster analysis indicated that the 16 varieties and two species formed one cluster. The first major cluster consisted of three varieties and a second major cluster consisted of two species and 13 varieties. The genetic distance was very close within the varieties and also among the species. Thus, these RAPD markers have the potential for identification of species/varieties and characterization of genetic variation within the varieties. This is also helpful in Hibiscus breeding programs and provides a major input into conservation biology

Genetic Variability and Relationships among Thirty Genotypes of Finger Millet (Eleusine coracana L. Gaertn.) Using RAPD Markers

Ragi or finger millet (Eleusine coracana L.) is an important crop used for food, forage, and industrial products. It is distributed in tropical and temperate regions of the world. The germplasm identification and characterization is an important link between the conservation and utilization of plant genetic resources. Traditionally, species or varieties identification has relied on morphological characters like growth habit, leaf architecture or floral morphology. Investigation through RAPD (random amplified polymorphic DNA) markers was undertaken for identification and determination of the genetic variation among thirty genotypes of ragi of the family Poaceae. Thirteen selected decamer primers were used for genetic analysis. A total of 124 distinct DNA fragments ranging from 300-3000 bp was amplified by using selected random RAPD marker. The genetic similarity was evaluated on the basis of the presence or absence of bands. Cluster analysis was made by the similarity coefficient. It indicated that the 30 genotypes of ragi form two major clusters, first, a major cluster having only one genotype, i. e. Dibyasinha and a second major cluster having twenty-nine genotypes. The second major cluster again subdivides into two minor clusters. A first minor cluster has only three varieties, i. e. Neelachal, OEB-56 and Chilika. The genotypes Neelachal and OEB- 56 exhibit a 86% similarity with each other and 80% similarity with Chilika. A second minor cluster has 26 genotypes and is divided into two sub-minor clusters. The first sub-minor cluster has only one genotype (VL-322). The second sub-minor cluster again subdivides into two groups. One group has one genotype and the second group again is divided into two sub-groups, one with 13 genotypes and the other with 11 genotypes. The highest similarity coefficient was detected in a genotype collected from southern India and the least from northern India. The genotypes of finger millet collected from diverse agroclimatic regions of India constitute a wide genetic base. This is helpful in breeding programs and a major input into conservation biology of cereal crop.

Genotypic Variability and Correlation Studies in Pod and Seed Characteristics of Pongamia pinnata (L.) Pierre in Orissa, India

Pongamia pinnata (L.) Pierre is a fast growing leguminous tree with high potential for oil seed production. Fifty-three candidate plus trees (CPTs) of Pongamia pinnata were selected from different locations in Orissa, India, on the basis of their seed and pod characteristics to identify suitable seed source with high oil content for production of quality planting seedling for use in afforestation programs. All the CPTs showed significant variation among themselves in respect to their pod and seed characters. Phenotypic coefficient of variation (PCV) and genotypic coefficient of variation (GCV) estimates were high for pod thickness, seed thickness, 100-pod weight, and 100-seed weight. High heritability values accompanied by high genetic advance for 100-seed weight (96.1%, 59.6) and 100-pod weight (90.9%, 37.3) indicated additive gene action. High estimates of genotypic correlations than the corresponding phenotypic correlations indicated the presence of strong inherent association between pod length and pod breadth; 100-pod weight, and pod thickness; 100-pod weight and seed length; 100-seed weight and 100-pod weight. Seed length, seed breadth, seed thickness, 100-pod weight and 100-seed weight had significant positive correlation with each other, and these characters should be considered as effective parameters to select CPTs for different agroforestry programs.

Genetic relationships among 23 Ficus accessions using inter-simple sequence repeat markers

Ficus genus is widely distributed in all the climatic stages and great diversity. The exploration of genetic diversity is a pre-requisite for genome organization in the wild species and the related domesticated ones. Inter-Simple Sequence Repeat (ISSR) markers were used to assess the identification of 23 important Ficus accessions and determination of the genetic relationships among these accessions. Out of 21 ISSR primers tested, five primers produced 116 detectable fragments, of which 106 (91.3%) were polymorphic across the accessions. Each of the five primers produced a fingerprint profile unique to each of the accessions studied, and thus could be solely used for their identification. Thirteen unique bands specific to nine species were detected. These may be converted into species-specific probes for identification purposes. Genetic relationships among these accessions were evaluated by generating a similarity matrix based on the Dice coefficient and the Unweighted Pair Group Method with Arithmetic Average (UPGMA) dendogram. The results showed a clear cut separation of the 23 Ficus accessions and were in broad agreement with the morphology. Both molecular and morphological markers will be useful for preservation of the Ficus germplasm.

Overexpression of a Barley Aquaporin Gene, HvPIP2;5 Confers Salt and Osmotic Stress Tolerance in Yeast and Plants

We characterized an aquaporin gene HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, Δ1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.

Identification of Variety Diagnostic Molecular Marker of High Yielding Rice Varieties

Twenty eight inter simple sequence repeat (ISSR) markers were used to determine the genotypic identification and phylogenetic relationship within 48 varieties of Indica rice including four aromatic rice grown in eastern part of India. Out of 44 upland and low land varieties, 39 varieties released by the Orissa University of Agriculture and Technology, Orissa and five released by the other National Institutes of India. A total of 28 ISSR primers were screened representing di- and tri- nucleotide repeats, out of which 12 ISSR markers were selected for varietal analysis as diagnostic markers. The number of bands per locus ranged from 4 to 8, with an average of 6. The results revealed that all the primers showed distinct polymorphism among the varieties indicating the robust nature of ISSR markers. Most of the primers showed highest polymorphic information content and resolving power. The cluster analysis indicates that all the rice genotypes are grouped into two classes in which aromatic rice varieties clustered separately from upland and lowland varieties. Principal component analysis showed the distinct differences among themselves. Based on this study, the larger range of similarity values using ISSR markers provides greater confidence for the assessment of genetic relationships among the varieties. The information obtained from the DNA profile helps to identify the variety diagnostic markers in 48 rice genotypes. This outcome of the investigation can be used in background selections during backcross breeding programs.

Identifi cation of Species-Diagnostic Inter Simple Sequence Repeat Markers for Ten Phyllanthus Species

Phyllanthus has been widely used in traditional medicine as an antipyretic, a diuretic, and to treat liver diseases and viral infections. Correct genotype identification of medicinal plant material remains important for the botanical drug industry. Limitations of chemical and morphological approaches for authentication have generated the need for newer methods in quality control of botanicals. In the present study, attempts were made to identify species- diagnostic markers for ten Phyllanthus species using the inter simple sequence repeat-polymerase chain reaction (ISSR-PCR) fingerprinting method. PCR amplification using seven ISSR primers resulted in significant polymorphism among the populations from different species. P. angustifolius and P. urinaria showed monomorphic frequency of maximum (63.88%) and minimum (20.64%), respectively. Seventeen species-diagnostic markers were identified for seven species (P. acidus, P. emblica, P. fraternus, P. urinaria, P. rotundifolius, P. amarus, and P. angustifolius) while no marker was detected for P. reticulatus, P. nivosus, and P. virgulatus. A maximum of six species-diagnostic markers were identified for P. acidus and a minimum of only one of 755 bp was available for P. amarus. Among the seventeen markers, nine were present in all individuals of particular species. The speciesspecific differences in fragment numbers and sizes could be used as diagnostic markers to distinguish the Phyllanthus species quickly

Stress Tolerance in Plants: A Proteomics Approach

Both abiotic and biotic stresses adversely affect the plant growth and productivity including crop plants. The development of stress-tolerant plants will be greatly advantageous for modern agriculture in areas that are prone to such stresses. In recent times, several advances have been made towards identifying potential stress-related genes which are capable of increasing the tolerance of plants to both abiotic and biotic stresses. This interaction between biotic and abiotic stresses is controlled by hormone signaling pathways that may induce or antagonize one another, in particular that of abscisic acid. Specificity in multiple stress responses is further controlled by a range of biochemical and molecular mechanisms that act together in a complex regulatory network. Transcription factors, kinase cascades, and reactive oxygen species are key components of this cross talk as are heat shock factors and small RNAs. This review emphasis on elucidating the proteins associated with abiotic and biotic stresses in plants.