Genome-wide analysis of ARF transcription factors reveals HcARF5 expression profile associated with the biosynthesis of ß-ocimene synthase in Hedychium coronarium.

KEY MESSAGE: Herein, 37 ARF genes were identified and analyzed in Hedychium coronarium and HcARF5 showed a potential role in the regulation of HcTPS3. Auxin is an important plant hormone, implicated in various aspects of plant growth and development processes especially in the biosynthesis of various secondary metabolites. Auxin response factors (ARF) belong to the transcription factors (TFs) gene family and play a crucial role in transcriptional activation/repression of auxin-responsive genes by directly binding to their promoter region. Nevertheless, whether ARF genes are involved in the regulatory mechanism of volatile compounds in flowering plants is largely unknown. ß-ocimene is a key floral volatile compound synthesized by terpene synthase 3 (HcTPS3) in Hedychium coronarium. A comprehensive analysis of H. coronarium genome reveals 37 candidate ARF genes in the whole genome. Tissue-specific expression patterns of HcARFs family members were assessed using available transcriptome data. Among them, HcARF5 showed a higher expression level in flowers, and significantly correlated with the key structural ß-ocimene synthesis gene (HcTPS3). Furthermore, transcript levels of both genes were associated with the flower development. Under hormone treatments, the response of HcARF5 and HcTPS3, and the emission level of ß-ocimene contents were evaluated. Subcellular and transcriptional activity assay showed that HcARF5 localizes to the nucleus and possesses transcriptional activity. Yeast one-hybrid (Y1H) and dual-luciferase assays revealed that HcARF5 directly regulates the transcriptional activity of HcTPS3. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays showed that HcARF5 interacts with scent-related HcIAA4, HcIAA6, and HcMYB1 in vivo. Overall, these results indicate that HcARF5 is potentially involved in the regulation of ß-ocimene synthesis in H. coronarium.

Secondary metabolites content and essential oil composition of in vitro cultures of Zingiber montanum (Koenig) Link ex A. Dietr.

OBJECTIVES: To determine the secondary metabolite content, antioxidant and phenylalanine ammonia-lyase (PAL) activity as well as essential oil composition of in vitro cultures and field grown rhizomes of Zingiber montanum. RESULTS: Methyl jasmonate-treated cell cultures showed the highest total phenolic content and steroid content of 22.23 mg gallic acid equivalent/g dry weight (DW) and 41.67 mg/g DW, respectively. Callus cultures exhibited the highest tannin content (39.53 mg tannic acid equivalent/g DW) and strongest antioxidant activity (91.05% inhibition of 2,2-Diphenyl-1-picrylhydrazyl or DPPH). The highest saponin (81.76 mg/g DW) and alkaloid (113.97 mg/g DW) contents were obtained in in vitro microrhizomes induced on Murashige and Skoog (MS) medium supplemented with 6% sucrose and 5 mg/l 6-Benzylaminopurine (BAP), and MS medium supplemented with 7% sucrose, respectively. The essential oil content varied in cell cultures and microrhizomes and mainly consisted of fatty acid esters, which are precursors of many secondary metabolites. Trace amounts of terpinen-4-ol (0.21 and 0.27 mg/g) and zerumbone (0.0107 mg/g) were also detected in the in vitro microrhizomes. CONCLUSION: The results obtained indicate the potential of in vitro cultures of Z. montanum for the production of secondary metabolites.

Effects of plant growth substances on rooting of Hedychium spicatum under different temperature regimes.

Present study was carried out to develop a simple and efficient vegetative propagation protocol by applying various treatments to rhizome cuttings with different test solutions of auxins and phenolic compound. These were alpha-naphthalene acetic acid (NAA), Indole-3-butyric acid (IBA), Indole Acetic Acid (IAA), phloroglucinol and coumarin. The concentrations for each treatment were 10.0, 50.0 and 100.0 microM. After treatments the rhizome cuttings were planted in polybags containing forest soil and kept under different temperature regimes i.e., inside polyhose (at 20-25 degrees C), inside mist chamber (at 15-20 degrees C) and under nethouse (nursery condition, at 14-18 degrees C). The maximum rooting percentage (74.06%) was achieved at 20-25 degrees C (inside polyhouse) by applying 50.0 microM IBA. Inside poly house condition, the various developmental parameters showed better responses compare to other conditions. On the basis of present study emphasizes that the temperature play a crucial role in rooting and further growth of the plants in this species. By using this simple and significant conventional method of propagation we could be propagate this vulnerable medicinal and aromatic species at large scale for commercial purpose.

Studies on antioxidant enzymes in Canna indica plant under copper stress.

Bright red-flowered Canna indica L. plants were subjected to grow in nutrient solution supplemented with five different concentrations (0, 5, 10, 30 and 50 microM) of CuCl2 to study antioxidant defense responses of the plant. Accumulation of Cu was dose-dependent and much higher in the roots (108-191 microg g(-1) d. wt.) than in the leaves (23.36-40.43 microg g d.wt.). Total ascorbate content did not changed in both tissues, but ascorbate redox state decreased (0.570-0.640) in Cu-treated Canna roots. In contrast, both total and reduced glutathione contents increased (387-591.9 nmol g(-1) f. wt.) considerably in roots, accompanied with enhanced activities of dehydroascorbate reductase (153.3-160 nmol mg(-1) protein) and glutathione reductase (67-87.5 nmol mg(-1) protein). No significant change, however, was observed for monodehydroascorbate reductase activity in both tissues of the treated plant. The efficient scavenging of hydrogen peroxide was performed by normal (control level) activities of both ascorbate peroxidase and catalase in leaf and increased activity of only catalase in root, preventing its accumulation at toxic concentrations (despite high superoxide dismutase activity) and subsequent damage of membrane lipids by peroxidation. Together, these ensured normal dry weight of leaves and roots, indicating tolerance of Canna indica plant to Cu-induced oxidative stress.

Induction of somatic embryogenesis in endangered butterfly ginger Hedychium coronarium J. Koenig.

An efficient protocol has been developed for regeneration of complete plants through somatic embryogenesis in H. coronarium. Creamish white, pale yellow and brown calli were obtained on MS medium supplemented with different concentrations of auxins [2, 4-Dichlorophenoxy acetic acid (2, 4-D), Indole-3 acetic acid (IAA) and 1-Naphthylacetic acid (NAA)] after 4 weeks. Creamy white calli developed on 0.5 mg L(-1) 2, 4-D turned embryogenic when subcultured on basal medium and produced small globular somatic embryos after 6 weeks. Further growth of somatic embryos required their transfer to medium containing 6-benzylaminopurine (BAP) or kinetin (KN). BAP was more effective than KN in promoting shoot proliferation. Maximum shoot length was obtained with 0.5 mg L(-1) BAP whereas maximum shoot number was obtained with 1.0 mg L(-1) BAP. The plantlets thus formed were successfully hardened, and transferred to sand-soil and farm yard manure (1:1:1) with 95% survival.

In vitro multiplication in Kaempferia galanga linn.

Kaempferia galanga is an important medicinal plant that is facing threat of extinction owing to indiscriminate and unsustainable harvesting in the wild. Conventional breeding is difficult in this plant, and in vitro multiplication is important to conservation and propagation. Leaf and rhizome explants of Kaempferia were aseptically cultured on MS medium with various combinations of indole-3-acetic acid (IAA), benzyl amino purine (BAP), napthalene acetic acid (NAA), 2-4-dichlorophenoxy acetic acid (2,4-D) and kinetin at concentrations ranging from 0.5 to 2.5 mg/L. High-frequency organogenesis and multiple shoot regeneration was induced from rhizome explants on MS medium supplemented with 0.5 mg/L of IAA and 2.5 mg/L of BAP. Rooting was induced in MS medium with 0.5 mg/L of IAA and 2 mg/L of BAP.