Subramani Rajkumar

Expressed sequence tags and molecular cloning and characterization of gene encoding pinoresinol/lariciresinol reductase from Podophyllum hexandrum

Podophyllotoxin, an aryltetralin lignan, is the source of important anticancer drugs etoposide, teniposide, and etopophos. Roots/rhizome of Podophyllum hexandrum form one of the most important sources of podophyllotoxin. In order to understand genes involved in podophyllotoxin biosynthesis, two suppression subtractive hybridization libraries were synthesized, one each from root/rhizome and leaves using high and low podophyllotoxin-producing plants of P. hexandrum. Sequencing of clones identified a total of 1,141 Expressed Sequence Tags (ESTs) resulting in 354 unique ESTs. Several unique ESTs showed sequence similarity to the genes involved in metabolism, stress/defense responses, and signalling pathways. A few ESTs also showed high sequence similarity with genes which were shown to be involved in podophyllotoxin biosynthesis in other plant species such as pinoresinol/lariciresinol reductase. A full length coding sequence of pinoresinol/lariciresinol reductase (PLR) has been cloned from P. hexandrum which was found to encode protein with 311 amino acids and show sequence similarity with PLR from Forsythia intermedia and Linum spp. Spatial and stress-inducible expression pattern of PhPLR and other known genes of podophyllotoxin biosynthesis, secoisolariciresinol dehydrogenase (PhSDH), and dirigent protein oxidase (PhDPO) have been studied. All the three genes showed wounding and methyl jasmonate-inducible expression pattern. The present work would form a basis for further studies to understand genomics of podophyllotoxin biosynthesis in P. hexandrum.

Catechin and catechin fractions as biochemical markers to study the diversity of Indian tea (Camellia sinensis (L.) O. Kuntze) germplasm.

The heterogeneous Indian tea germplasm includes ‘China’, ‘Assam’, ‘Cambod’, and their hybrids which were evaluated using biochemical markers viz., total catechin and their fractions, for varietal identification and characterization. Principal component analysis (PCA) of biochemical characters showed that the total catechin and trihydroxylated catechin has higher eigenvalues. The first two principal components (PCs) could differentiate more than 90% of the clones studied. This grouping based on first two principal component matrices differentiated ‘China’, and their hybrids with ‘Assam’ and ‘Cambod’ variety. Morphologically indistinct large‐leaved ‘Cambod’ variety and ‘Assam’ varieties could not be differentiated using biochemical markers, since both varietal types taxonomically belong to a single species. Clones of ‘China’ type showed low total catechin content and catechin ratio which are distinctly grouped. The ‘China–Assam’ and ‘China–Cambod’ hybrids formed intermediate groups between ‘China’ PC group and ‘Cambod’/‘Assam’ PC groups, providing evidence for genetic control of catechin ratio variation. Tea clones which are differentially positioned in the PC group could be explained based on the genetic contribution by other varietal type as parents. This biochemical characterization will be a useful tool in the development of quality‐tea clones with different proportion of total catechin and their fractions.

High Level of Genetic Diversity Among the Selected Accessions of Tea (Camellia sinensis) from Abandoned Tea Gardens in Western Himalaya

To revive cultivation of the tea unique to the western Himalayan region, it is important to evaluate the seed-derived bushes available in the area’s abandoned gardens. This study used quantitative leaf characters, catechin content, and AFLP markers to assess these China cultivar type bushes. Compared with other China cultivar germplasm, these accessions showed a higher level of diversity among themselves. Among the quantitative morphological characters, leaf length is important in distinguishing the accessions studied, with a high loading value in the principal component analysis. The catechins and AFLP markers displayed the genetic makeup of the accessions. Other than total catechins, the trihydroxylated catechins showed a high loading value in differentiating the accessions. The genetic control of the ratio of dihydroxylated and trihydroxylated catechins is found to be based on a correlation with AFLP markers. The genetic similarity between Kangra Asha and Kangra Jat suggests that Kangra Jat must be descended from Kangra Asha. Kangra Jat is well adapted to local environmental conditions, as is evident from its high catechin content.

Bacterial diversity and soil enzyme activity in diseased and disease free apple rhizosphere soils

A culture-independent survey of the bacterial diversity in rhizosphere soils of diseased (scab) and disease-free apple trees was conducted to assess the role of bacteria in disease suppression. Community DNA was extracted from soil samples and amplified by PCR using primers specific for bacterial 16S rRNA gene sequences. Clone libraries were constructed with the PCR products and analysed based on amplified rDNA restriction analysis (ARDRA) patterns. The phylotypes and their frequency distribution in both libraries indicated that the phylotype did not represent a single group. Rarefaction curve, and Shannon and Simpson diversity indices exhibited insignificant diversity differences between the samples in terms of bacterial community composition, whereas more chitinase and β-1,3 glucanase activities were recorded in samples from disease-free trees than from diseased trees. Based on the operational taxonomic units identified in ARDRA, 80 representative clones were selected from the libraries and partially sequenced. Sequence similarity searches with the resulting sequences identified the dominance of uncultured bacteria to the extent of 70% and 72.5% in disease free and diseased rhizospheres, respectively. The foregoing studies conclude a possible role for enhanced microbial activity in terms of enzyme production in tree (apple) health, although no distinct partitioning of composition or significant diversity of bacterial communities inhabiting diseased and disease-free rhizosphere soils was observed at any given time.