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This is the first report on de novo transcriptome of Dactylorhiza hatagirea, a critically-endangered, terrestrial orchid of alpine Himalayas. The plant is acclaimed for medicinal properties but little is known about its secondary-metabolites profile or cues regulating their biosynthesis. De novo transcriptome analysis was therefore, undertaken to gain basic understanding on these aspects, while circumventing the acute limitation of plant material availability. 65,384 transcripts and finally, 37,371 unigenes were assembled de novo from a total of 236 million reads obtained from shoot, tuber and leaves of the plant. Dominance of differentially-expressing-genes (DEGs) related to cold-stress-response and plant-hormone-signal-transduction; and those involved in photosynthesis, sugar-metabolism and secondary-metabolite-synthesis provided insights into carbohydrate-partitioning in the plant during its preparation for freezing winter at natural habitat. DEGs of glucomannan, ascorbic acid, carotenoids, phylloquinone/naphthoquinones, indole alkaloids, resveratrol and stilbene biosynthesis revealed the secondary-metabolite profile of D. hatagirea. UHPLC results confirmed appreciable amounts of resveratrol and trans-stilbene in D. hatagirea tubers, for the first time. Expression analysis of 15 selected genes including those of phenylpropanoid pathway confirmed the validity of RNA-seq data. Opportunistic growth, temperature- and tissue-specific-differential-expression of secondary metabolite biosynthesis and stress tolerant genes were confirmed using clonal plants growing at 8, 15 and 25 °C.

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The study is the first report on de novo transcriptome analysis of Nardostachys jatamansi, a critically endangered medicinal plant of alpine Himalayas. Illumina GAIIx sequencing of plants collected during end of vegetative growth (August) yielded 48,411 unigenes. 74.45% of these were annotated using UNIPROT. GO enrichment analysis, KEGG pathways and PPI network indicated simultaneous utilization of leaf photosynthates for flowering, rhizome fortification, stress response and tissue-specific secondary metabolites biosynthesis. Among the secondary metabolite biosynthesis genes, terpenoids were predominant. UPLC-PDA analysis of in vitro plants revealed temperature-dependent, tissue-specific differential distribution of various phenolics. Thus, as compared to 25 °C, the phenolic contents of both leaves (gallic acid and rutin) and roots (p-coumaric acid and cinnamic acid) were higher at 15 °C. These phenolics accounted for the therapeutic properties reported in the plant. In qRT-PCR of in vitro plants, secondary metabolite biosynthesis pathway genes showed higher expression at 15 °C and 14 h/10 h photoperiod (conditions representing end of vegetative growth period). This provided cues for in vitro modulation of identified secondary metabolites. Such modulation of secondary metabolites in in vitro systems can eliminate the need for uprooting N. jatamansi from wild. Hence, the study is a step towards effective conservation of the plant.

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The PDS1000-He biolistic gun was used to bombard plasmid DNA harbouring gus and nptII genes into tender young leaves of in vitro grown shoots of Camellia sinensis (tea). Out of a total of 500 bombarded leaves, 217 (43.4 %) showed callusing after 5 weeks on selection medium containing 1.71 lM kanamycin. Only 15 of these regenerated into indirect shoot buds. Only 7 out of 15 putative transformants showed the expected 400 bp signal with gus gene specific primers during PCR analysis. On the other hand, all the 15 putative transformants tested positive with nptII gene specific primers. In Southern hybridization with nptII specific gene probe, all the six randomly selected PCR positive plants showed stable integration of nptII gene. Both the transgenic and not-bombarded control plants showed phenotypic similarity under polyhouse conditions. Although their growth parameters were significantly at par, significantly lesser shoot height was recorded in transgenic plants. The reproductive behavior of the transgenics was also depressed. Thus, floral bud and flower abscission, fruit drop as well as empty seed production was higher in the transgenics as compared to control. Viability and germination of transgenic seeds was also significantly lower than control. Survival of the transgenic seedlings was also negligible (about 3 %). Hence, the chances of germline transmission of the transgenes were remarkably reduced in case of gus-transgenics. Tea being a vegetatively propagated plant, the method described in the present paper is an important approach for developing transgenics of elite tea plants from leaf explants.

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