K. Baskaran

Higher efficiency of ISSR markers over plastid psbA‐trnH region in resolving taxonomical status of genus Ocimum L.

Abstract High level of morphological as well as chemical variability exists within the genus Ocimum, and its taxonomy and phylogenetic relationships are still doubtful. For evaluating interspecific genetic relationships among the Ocimum species, genotyping with intersimple sequence repeat (ISSR) markers and sequence analyses of noncoding psbA‐trnH intergenic region belonging to chloroplast DNA were carried out. Although ISSR markers are highly efficient and reproducible, they have not been used extensively in phylogenetic studies. The use of the plastidial barcode candidate was expected to provide more variable and informative insight into evolutionary rates, and was thus employed as a phylogenetic marker to assess interspecific relationships. This study revealed that the ISSR markers were more efficient than psbA‐trnH sequences in resolving the current status of Ocimum L. genus. Distance‐ and character‐based methodological approaches applied on the molecular data with biparental and maternal inheritance were used for deducing the phylogenetic relationships among Ocimum species. Average polymorphic information content (0.344) and resolving power (6.285) depicted through ISSR markers proved to be efficient in discriminating the studied species of Ocimum. The primers used in this study revealed 99.585% polymorphism across the species demonstrating the polymorphic nature of ISSR markers.

From Herkogamy to Cleistogamy - Development of Cleistogamy in Periwinkle

Periwinkle (Catharanthus roseus [L.] G. Don), an important medicinal plant, is an allogamous species in which the stigma is below the anthers. The receptive portion is at the base of the stigmatic head and thus automatic intra-flower self-pollination is excluded. The structure of the flower is of typical reverse herkogamy and pollination occurs through nectar-seeking insects. A few self-pollinating strains are also reported in which self-pollination is brought about by an increase in length of the style or of the ovary. Self-pollination is governed by allelic duplicate genes recessive to allogamy. An induced monogenic recessive mutant (EMS 17-1) with caducous closed corolla (corolla abscising before anthesis), isolated from variety, Dhawal, was crossed with two self-pollinating strains to study the possibility of obtaining cleistogamous recombinants combining closed corolla and self-pollination traits. Cleistogamous plants were obtained in which development of fruits and seeds occurred without opening of the corolla. Closed corolla and self-pollination were found to be independently inherited. A dominant gene in the parent in which self-pollination occurred due to an increase in length of the ovary, appeared to completely or partially inhibit expression of the gene for closed corolla in homozygous or heterozygous condition, respectively. The genetic basis of development of cleistogamy is described. Cleistogamy in periwinkle would facilitate in ensuring genetic purity, pollen containment, and seed production even in the absence of pollinators. This appears to be the first report on the development of cleistogamous plants in an allogamous species.

Genetics of novel corolla colours in periwinkle

Inheritance of a novel corolla colour in periwinkle [Catharanthus roseus (L) G. Don], viz. magenta, was studied by crossing an accession MJ, possessing this corolla colour, with cultivar Nirmal, possessing white corolla. The accession MJ was also crossed with another accession OR, possessing another novel corolla colour, viz. orange-red, to determine the relationship between genes governing magenta corolla and orange-red corolla. The F1 plants of the cross MJ× Nirmal had pink corolla and red eye. In the F2 generation, five kinds of corolla colours were observed: (i) pink corolla and red eye, (ii) rose corolla and red eye, (iii) magenta corolla and red eye, (iv) white corolla and red eye and (v) white corolla. The observed frequencies of the five kinds of plants fitted a ratio of 144:27:9:12:64. The progeny of the backcross, F1 × MJ, segregated into three kinds of plants, (i) pink corolla and red eye, (ii) rose corolla and red eye and (iii) magenta corolla and red eye, in the ratio of 2:1:1, while the backcross, F1 × Nirmal, segregated into two kinds of plants, (i) pink corolla and red eye and (ii) white corolla, in the ratio of 1:1. Two new genes (proposed symbols Om and J) appeared to be involved in the determination of magenta and rose corolla colours. Interaction between four independent genes R, W, Om and J, appeared to explain the observed segregation in the cross MJ × Nirmal. The F1 plants of the cross MJ × OR had scarlet-red corolla and red eye. The segregation data of F2 and backcross generations suggested that genes governing orange-red corolla and magenta corolla were allelic to each other. Two new and non-parental corolla colours viz., rose corolla and scarlet-red corolla, were observed in the progeny of the crosses of the present study.

ISSR-Derived Species-Specific SCAR Marker for Rapid and Accurate Authentication of Ocimum tenuiflorum L.

Ocimum tenuiflorum has been widely used in traditional medicine and has high medicinal value. High volume trade of this potential medicinal plant species led to unscrupulous adulteration of both crude drugs as well as formulations. Morphology-based authentication is difficult in cases of incomplete or damaged samples and in dried herbal materials. In such cases, PCR-based molecular methods may aid in accurate identification. The present study aimed at developing species-specific DNA marker(s) for the authentication of O. tenuiflorum. A species-specific amplicon (279 bp) generated through an inter-simple sequence repeat marker (UBC 835) in all individuals of O. tenuiflorum was cloned, sequenced, and a primer pair was developed (designated as CIM-OT-835F/CIM-OT-835R). The newly developed sequence characterized amplified region marker was validated through PCR amplification in all available seven species of Ocimum, and its specificity for O. tenuiflorum was confirmed with the consistent generation of an amplicon of 177 bp. The developed marker can be used for accurate and rapid identification of the species for certification purposes and will be useful in quality control of medicinal preparations containing this important medicinal species.

Individual and combined effects of genes producing opposite effects on plant height in periwinkle ( Catharanthus roseus )

An extremely tall mutant (EMS 18-12), a bushy mutant (EMS 24-5), their parental variety Nirmal, and their double mutant recombinant were used to study individual and combined effects of genes producing opposite effects on plant height in periwinkle (Catharanthus roseus). Plant height of the extremely tall mutant (EMS 18-12) was controlled by epistatic inhibitory interaction between two independently inherited dominant genes, Et and H; Et producing extremely tall phenotype and H inhibiting Et. Both genes were inherited independently of plant height-reducing recessive gene by in the bushy mutant (EMS 24-5). Individually, genes Et and by increased and reduced plant height at harvest (when plant were 9 months old) by 90 and 25%, respectively, over parental variety. The double mutant recombinant (Etby) was taller than the bushy mutant (EMS 24-5) and variety, Nirmal but shorter than the extremely tall mutant (EMS 18-12) at different stages. At 1 to 7 weeks after germination, its height was 7.4 to 30.0% greater than the mid-parental value but 5.8 to 30.5% shorter than that expected on the basis of individual effects of genes Et or by. At the age of 4 to 9 months, its height was 5.4 to 40.1% greater than the mid-parental value and 5.6 to 44.1% (except at 5 months) greater than that expected on the basis of individual effects of genes Et or by, suggesting age-dependent epistatic interaction between the genes. No interaction effects were observed for leaf and root yields or contents of alkaloids in leaves and roots.