Studies on in vitro propagation and screening of athocyanin and volatile compounds of vanda orchids

Vanda helvola belongs to the Borneo orchid and listed as one of the endangered orchid species. The in vitro propagations of V. helvola were successfully established through seed germination and leaf segment culture. The effect of basal media (KC, MS, and VW); complex additives (tomato juice, coconut water, yeast extract and peptone); and plant growth regulators (NM, BAP, 2, 4-0 and kinetin) were examined to determine the optimum medium for seed germination, protocorm and seedling growth, protocorm proliferation and PLBs induction from leaf culture. All cultures from the entire studies were maintained under 24 h light conditions at 25 ± 2°C. Seeds of V. helvola were aseptically cultured and germinated for over 90% on Knudson C (KC) basal medium supplemented with 10 - 15% (v/V) of tomato juice after 90 days of culture. The germinated seed developed to protocorm and the establishment of V. helvola seedling was best performed in medium added with 0.1 - 0.2% (wtv) of peptone. The final seedling stage was achieved with a formation of 4 - 5 leaves and 3 roots. The acclimatization of seedling was then achieved when the well-developed seedling was transferred to pot containing brick pieces and coconut husks (1:2) and mulched with Sphagnum sp. The potted plants survived with 55.67% after 12 months, and shifted to Taman Pertanian Lagud Sebrang for further growth. The proliferation of V. helvola protocorm was demonstrated on KC basal medium with addition of NAA or BAP. Multiplication of protocorm was high on KC basal medium containing 1 mgtl BAP with a production of 10.57 ± 0.36 new protocorms per responsive explant. The addition of 2.0 mgt I NAA to this treatment has lowered the proliferation rate (7.46 ± 0.36), but increased the shoot/leaf promoting effect of the new protocorms. The PlBs induction from leaf segment culture produced a range of 5 - 9 PLBs on Mitra basal medium supplemented with 0.5 mg/l NAA and 4.0 mg/l BAP after 90 days of culture. Besides the tissue culture study, a preliminary screening on anthocyanin and volatile content has been done on two Borneo Vanda species i.e., V. helvola and V. dearei. The results showed that a high PAL enzyme activity in V. dearei flower might concurrent with the present of cinnamaldehyde compound from GCMS analysis, which supported with a strong fragrance emission from this flower. Meanwhile, higher anthocyanin content in V. helvola flower extract might be related with the colour of this flower. The successful establishment of the tissue culture techniques on V. helvola would be beneficial to the Borneo orchid's conservation, while the preliminary data on anthocyanin pigmentation and volatile content might be useful for future research in understanding the regulation of these compounds

Organic additives improves the in Vitro growth of native Orchid Vanda helvola Blume

In vitro seed germination has been proven to be the most efficient technique to propagate orchid. The application of this aseptic technique has contributed to conservation of many endangered orchid species. In this study, undehisced capsules of Vanda helvola Blume were collected from Orchid Conservation Centre in Lagud Sebrang Agriculture Park, after 120 days from hand pollination and aseptically cultured on three types of basal media such as Murashige and Skoog (MS), Knudson C (KC) and Vacin and Went (VW). After 90 days of culture, 66.40 ± 4.14% of seeds successfully germinated on KC medium. The effect of organic additives such as tomato juice, coconut water, peptone and yeast extract at different level of concentrations in KC basal medium were also tested on seed germination and seedling development of this native orchid. After 90 days of culture, over 90% of seeds were tremendously germinated on KC medium supplemented with 10% or 15% (v/v) of tomato juice. The incorporation of peptone at 0.1% (w/v) in KC basal media promoted rapid development of protocorm to seedling. Seedlings on this treatment produced an average of three leaves and two roots after 90 days of culture and were successfully acclimatized

Asymbiotic seed germination and seedling development of Vanda dearei

The effects of basal media, complex additives, plant growth regulators and carbon sources on in vitro seed germination and seedling development of Vanda dearei are reported. Immature seeds from four months old capsule were used as plant materials. All cultures were grown under 24h light at 25±2ºC. Results showed that seeds cultured on Knudson C (KC) basal medium germinated after 25 days with 63.0±3.2% germination rate followed by half-strength Murashige & Skoog (½MS) (45.4±10.4%) and Vacin and Went (VW) (41.8±4.0%). Addition of 0.5% (w/v) yeast extract significantly enhanced (85.9±0.7%) seed germination and shortened germination time to 23 days. A NAA at 0.1mg/l had similar performance (80.2±20.5%), however, this treatment delayed seed germination and induced necrosis to protocorm development. Sucrose at 1% (w/v) also enhanced seed germination (98.3±2.3%), while glucose and fructose treatments showed moderate effects. For growth and development of protocorms, KC basal media recorded the highest percentage of protocorm with root (37.0±4.3%), mean number of leaf (4.50±1.00) and mean number of roots produced (2.0±0.6) with largest leaf area (3.7x2.3mm) and longest root length (11.7±8.4mm). Addition of 20% (v/v) coconut water significantly improved protocorm development and shoot growth

The origin of pulsating ultra-luminous X-ray sources: Low- and intermediate-mass X-ray binaries containing neutron star accretors

Ultra-luminous X-ray sources (ULXs) are those X-ray sources located away from the centre of their host galaxy with luminosities exceeding the Eddington limit of a stellar-mass black hole ($L_X>10^{39}\;{\rm erg\,s}^{-1}$). The discovery of X-ray pulsations in some of these objects (e.g. M82~X-2) suggests that a certain fraction of the ULX population may have a neutron star accretor. We present systematic modelling of low- and intermediate-mass X-ray binaries (LMXBs and IMXBs; donor-star mass range $0.92$--$8.0$~M$_{\odot}$ and neutron-star accretors) to explain the formation of this sub-population of ULXs. Using MESA, we explored the allowed initial parameter space of binary systems consisting of a neutron star and a low- or intermediate-mass donor star that could explain the observed properties of ULXs. Our simulations take into account beaming effects, stellar rotation, general angular momentum losses, and a detailed and self-consistent calculation of the mass-transfer rate. We study the conditions that lead to dynamical stability of these systems, which depends strongly on the response of the donor star to mass loss. Using two values for the initial neutron star mass ($1.3$~M$_{\odot}$ and $2.0$~M$_{\odot}$), we present two sets of mass-transfer calculation grids. We find that LMXBs/IMXBs can produce NS-ULXs with typical time-averaged isotropic-equivalent X-ray luminosities of $10^{39}$--$10^{41}\;{\rm erg\,s}^{-1}$ on a timescale up to $\sim\!1.0\;{\rm Myr}$ for the lower luminosities. We also estimate their likelihood of detection, the types of white-dwarf remnants left behind by the donors, and the total amount of mass accreted by the neutron stars. We also compare our results to the observed pulsating ULXs. Our results suggest that a large subset of the observed pulsating ULX population can be explained by LMXBs/IMXBs undergoing a super-Eddington mass-transfer phase.Comment: 19 pages, 13 figures, Accepted by A&A. Parameter space was increased to include low-mass XRBs and corresponding changes made to the text (including the title) and figures 4, 6-11. Changed axes for figures 1 and 2. Fixed typos and updated references. Added arguments about why spin period is not an accurate reflection of mass accretion rate in the introductio

Effects of Fertilizer Application and Successive Harvesting on Clipping Yield, Phytochemical Contents and Antioxidant Activity of Cynodon dactylon (L.) Pers.

The present study aimed to investigate the effects of fertilizer application and successive harvesting on phytochemical contents and antioxidant activities of Cynodon dactylon, a medicinal Bermuda grass in Sabah (Malaysia). Three fertilizers of two nitrogen concentrations were used in the experiment. The grass was harvested successively three times at five-weeks interval. Grass treated with 25 kg N/ha/month from the first harvest was found to have the highest clipping yield. Successive harvesting decreased the dry matter production of the grass irrespective of N concentration applied. Total saponin and alkaloid contents of the grass were increased by a combination treatment of fertilizer type ´ rate ´ harvesting; total flavonoid content was increased by fertilizer type ´ harvesting treatments; however, total phenolic content was not affected by any of the treatment or combination of the treatments. Both of the antioxidant assays (DPPH and FRAP) indicated that antioxidant activity of the grass was increased by fertilizer rate ´ harvesting treatments. There was a significant correlation found between total phenolic and flavonoid contents and antioxidant activities, suggesting that these two secondary metabolites may contribute to the antioxidant property of the grass. Overall, the obtained data indicated that the described treatments could be used to manipulate the production and accumulation of bioactive compounds of C. dactylon

In vitro seed germination of Coelogyne asperata Lindl. (Orchidaceae)

A protocol for in vitro seed germination of Coelogyne asperata Lindl. has been established successfully. Immature seeds from 182 days old capsule were cultured on three different basal media; Murashige and Skoog (MS), Knudson C (KC), and Vacin and Went (VW) and maintained under continuous light at 25 ± 2 ˚C. After 30 days of culture (DAC), more than 90% of seeds were germinated on KC and VW media and about 84% of seeds germinated on MS medium. The incorporation of organic additives, including coconut water, potato homogenate and tomato juice each at 10% (v/v) in KC medium was tested to determine their effect on seed germination of C. asperata. The result revealed that KC basal medium alone without addition of organic additives promoted over 90% of seed germination at 30 DAC. Therefore, the protocol of using standard KC basal medium for C. asperata seeds germination could be suggested for mass propagation and conservation of this wild scented orchid

Effects of Fertilizer Application and Successive Harvesting on Clipping Yield, Phytochemical Contents and Antioxidant Activity of Cynodon dactylon (L.) Pers.

The present study aimed to investigate the effects of fertilizer application and successive harvesting on phytochemical contents and antioxidant activities of Cynodon dactylon, a medicinal Bermuda grass in Sabah (Malaysia). Three fertilizers of two nitrogen concentrations were used in the experiment. The grass was harvested successively three times at five-weeks interval. Grass treated with 25 kg N/ha/month from the first harvest was found to have the highest clipping yield. Successive harvesting decreased the dry matter production of the grass irrespective of N concentration applied. Total saponin and alkaloid contents of the grass were increased by a combination treatment of fertilizer type ´ rate ´ harvesting; total flavonoid content was increased by fertilizer type ´ harvesting treatments; however, total phenolic content was not affected by any of the treatment or combination of the treatments. Both of the antioxidant assays (DPPH and FRAP) indicated that antioxidant activity of the grass was increased by fertilizer rate ´ harvesting treatments. There was a significant correlation found between total phenolic and flavonoid contents and antioxidant activities, suggesting that these two secondary metabolites may contribute to the antioxidant property of the grass. Overall, the obtained data indicated that the described treatments could be used to manipulate the production and accumulation of bioactive compounds of C. dactylon

In vitro propagation of Zingiber officinale Rosc. ‘Tambunan’

Rhizome buds of ginger (Zingiber officinale Rosc. ‘Tambunan’) were sterilized and cultured on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of NAA and BAP hormones (1-3 mg/L) to induce shoot multiplication and rooting formation. Shoot formation was first observed on treatment of 3.0 mg/L BAP + 1.0 mg/L NAA after 7 days of culture. This treatment also promote the highest number of proliferated shoots, 6.14 ± 0.91 shootlets per explant, with an average shoot length of 1.69 ± 0.17 cm observed after 10 weeks of culture. Rooting of ginger plantlets were significantly initiated on medium supplemented with 2.0 mg/L NAA. This treatment induced up to 34.40 ± 1.81 roots per explant with an average length of 4.52 ± 0.20 cm after 10 weeks of culture. Plantlets were successfully acclimatized in pot containing medium mixture of sand and clay (1:4) with 64% of survivality after transplanted for 3 weeks

Optimizing sucrose and BAP concentrations for in vitro microrhizome induction of Zingiber officinale Rosc. ‘Tambunan’

In vitro microrhizome induction is considered as an effective tool for high yielding rhizomatous crops. In this study, the effect of sucrose and BAP was examined to establish a suitable protocol for in vitro microrhizome production of Zingiber officinale Rosc. ‘Tambunan’. The in vitro derived plantlets were used as explants and cultured on Murashige and Skoog (MS) medium treated with a combination of sucrose and BAP at various concentrations and maintained at 25 ± 2°C with 16 hr of photoperiod. After three months of culture, explant responded well on MS medium supplemented with 60 g/L of sucrose and 6 mg/L of BAP compared to other treatments. This treatment had significantly promoted the highest number of microrhizomes (seven) with a total weight of 2.90 g and a total number of 35 buds. Acclimatization of this microrhizome showed 88% of survivability rate after 21 days with a formation of new shoot and root. The current finding revealed the potential of microrhizomes for large-scale production of healthy planting material to support the ginger industry in this region

Effect of NAA and BAP on protocorm proliferation of Borneo scented orchid, Vanda helvola

A simple and efficient multiplication protocol was develop for Vanda helvola using protocorms produced by seeds germination as explant sources. Protocorms were cultured on Knudson C as a basal medium with addition of alpha-Naphtaleneacetic acid (NAA) or 6-benzylaminopurine (BAP) singly or combinations under continuous illumination at 25±2ºC for 180 days after culture (DAC). In single hormone study, protocorms were cultured on media supplemented with NAA or BAP (0.1, 0.5, 1.0, 2.0 and 4.0 mg l-1). At 120 DAC, treatments with NAA or BAP (0.5, 1.0 and 2.0 mg l-1) gave an obvious multiplication rate and were proceed to the next study of hormones combinations. All protocorms were proliferated on all treatments at 180 DAC but varied in number of new protocorms they produced and the time required for the initiation of new protocorms. Protocorms cultured on a medium containing 2.0 mg l-1 BAP + 0.5 mg l-1 NAA produced the highest percentage of new protocorms (65 ± 20.88%) with an average of 3 ± 0.77 per protocorm at 60 DAC. Production and development of new protocorms increase until 180 DAC with an average 9 ± 2.2 new protocorms produced per explant. First shoot (0.5-1.0 mm) were observed on new protocorms at 60 DAC. After 180 days of culture new protocorms produced two leaves (2-3 mm in length)