Phylogenetic relationships of Indian Memecylon L. (Melastomataceae) based on nrDNA ITS and cpDNA rbcL sequence data.

Memecylon (Melastomaceae) is a large genus of the Old-World predominantly woody species. Many species of Memecylon are used for timber, ornamental and medicinal purposes. The objective of the present study was to undertake a phylogenetic analysis of Indian Memecylon based on nuclear ribosomal DNA internal transcribed spacer (nrDNA-ITS) and rbcL sequence data. Sampling included 26 species and one variety (20 endemics) representing 67% of the total Indian species. Molecular phylogeny data for analysed species revealed that the Indian Memecylon is monophyletic. Monophyly is strongly supported in the ITS, rbcL and ITS + rbcL combined analyses. Memecylon species are grouped in a major clade with strong support in ITS sequence data and moderate support in combined ITS + rbcL analyses.

Ultrastructural studies and molecular characterization of root-associated fungi, of Crepidium acuminatum (D.Don) Szlach.: a threatened and medicinally important taxon.

Crepidium acuminatum (Orchidaceae) is a threatened medicinal orchid that grows under shady and moist forest floor where light remains for a very short period of time. Mycorrhizal association is known to be essential for seed germination and seedling establishment in amajority of orchids. Identification of fungi that form mycorrhizae with orchids is of crucial importance for orchid conservation. We used both morphological as well as molecular approaches to study this plant-fungal interaction. Scanning electron microscopy showed that fungi grow and proliferate in the middle layers of the cortex. Also, spiral-root hairs were foundalong with root hairs, which is an unusual observation. Spiral-root hairs provide more surface area for fluid absorption and entrance of colonizers. Further, total root genomic DNA was isolated and fungal internal-transcribed spacer (ITS) regions were polymerase chain reaction (PCR)-amplified using specific primer combinations ITS1F/ITS4 and ITS1/ITS4tul. ITS sequences were obtainedand analysed to know the closest sequence matche in the GenBank using BLASTn hosted by NLM-NCBI. Subject sequences were identified to be belonging to three main genera, namely, Tulasnella, Aspergillus and Penicillium. Results indicate that mycorrhizal association is necessary for the growth and development of the plant. In addition, this symbiosis influences the distribution and rarity of this medicinally valuable taxon. Specific fungal partners may lead to an enhanced seed germination rate and increased efficiency of nutrient exchange between both the partners. Hence, knowledge of mycorrhizal fungi is essential for future in vitro germination and seedling establishment programmes, because they rely on fungi for germination. Identification of mycorrhizal fungi can be usedfor orchid propagation and conservation programmes.

Phylogenetic analysis and evolution of morphological characters in the genus Jasminum L. (Oleaceae) in India.

Jasminum L. (Oleaceae) consists of ∼200 species that are distributed in tropical, subtropical and temperate regions of the world. In India, this genus is represented by ca 47 species of which 16 are endemic. Based on the nuclear (internal-transcribed spacer (ITS) region of nrDNA and chloroplast markers (matK, trnL-F and trnH-psbA), phylogenetic relationships in 22 species including one variety of Jasminum in India have been assessed. Maximum likelihood and Bayesian analyses from individual markers, as well as from combined dataset, reveal that the group is monophyletic if Menodora spp. are excluded from the analyses. Our analyses recovered three strongly supported clades. Ancestral character state reconstruction of taxonomically useful characters (leaf forms, leaf arrangement and flower colour) which were used to demarcate sections within the genus reveals homoplasy. Our study suggests that after split from the last common ancestor, there have been at least four reversals to unifoliolate condition. Pinnately compound leaf form evolved at least twice and trifoliolate condition evolved one time only. Alternate leaf form evolved at least twice, once inclade 1 and once in clade 3 and all the time from ancestors having opposite leaf forms. Flower colour evolution clearly depicts that clade 1 is yellow-flowered and clades 2 and 3 have admixture of white and yellow-flowered Jasminum species. Our study suggests that yellow-flowered condition evolved from the white-flowered ancestor. The present study is first to estimate the evolutionary history of Indian Jasmines.

Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant.

Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habitats. As raising plants through seeds in this plant is problematic, a reliable protocol for micropropagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plantlets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomorphic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respectively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocarpum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations.