Seeds of P. japonicum used in the present study were collected from Penghu Island in Taiwan (23°36′18.0″N 119°31′08.7″E). Commercial samples of P. japonicum from Japan and Taiwan were obtained from the authorized sources.
Establishment of aseptic seedlings
Seeds of P. japonicum were disinfected by washing several times with sterile distilled water, followed by dipping in 70 % ethanol (v/v) for 10 s, then immersing in a solution of 0.5 % (v/v) sodium hypochlorite containing 1 drop of Tween-20 for 5 min and the step repeated three times. Final washing step carried out in a laminar flow cabinet consisted of 3 rinses of 5 min each with sterile distilled water. Thereafter, disinfected seeds were inoculated in pre-sterilized petridishes (90 mm dia). Each dish contained 20 mL of 1× Murashige and Skoog’s (1962) salts and vitamins, hereinafter referred as MS basal medium (MSBM). Gellan Gum powder (GPP) (0.4 %) purchased from PhytoTechnology Laboratories® was used as a gelling agent and 3 % sucrose was supplemented to each medium. The pH of all the media was adjusted to 5.7 ± 0.1, prior addition of GPP, and before autoclaving for 15 min under 1.05 kg/cm at 121 °C. The petridishes were incubated in a culture room at 25 ± 2 °C, a light and dark cycle of 16/8 h and an illumination intensity of 34 μmol/m2/s. After 14 days, germinated seeds were transferred to glass bottles (650 mL capacity) each containing 100 mL of MS basal medium supplemented with 3 % sucrose and 0.4 % GPP.
Induction of callus
For induction of somatic embryogenesis in P. japonicum, initial experiments were carried out to find out a suitable culture medium and an explant. Root, leaf blade and petiole parts of 35 days old in vitro raised seedlings of P. japonicum were used as explants. These were separately inoculated in pre-sterilized petridishes (90 mm). Each petridish contained 20 mL of MS basal medium supplemented with a range of concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) (0.1–5.0 mg/L). Each medium was supplemented and 3 % sucrose and 0.4 % GPP. Cultures were incubated in a culture room at 25 ± 2 °C in dark. After 35 days, induction of callus in each was recorded. Calli induced were subcultured for three cycles, on MSBM with same 2,4-D concentration. At the end of each subculture cycle of 35 days, fresh weight and morphological changes in the calli were recorded. Out of three explants, root showed the maximum callus proliferation, hence root callus was used for further experiments for induction of somatic embryos.
Influence of ABA on induction of somatic embryos
To investigate the influence of ABA on induction of somatic embryos, callus was cultured in petridishes (90 mm) containing MS basal medium with abscisic acid (ABA) (0.5, 1.0, 2.0, 4.0 mg/L), 3 % sucrose and 0.4 % GPP. Five clumps of 100 mg callus was inoculated in each petridish. These were incubated in a culture room at 25 ± 2 °C in dark. After 60 days of incubation, morphological changes and induction of somatic embryos in the calli were recorded.
Influence of different light spectra on induction of somatic embryos and callus proliferation
This experiment was performed to evaluate the influence of different light spectra on induction of somatic embryos, proliferation of callus (fresh weight) and conversion of somatic embryos into plantlets. Also, the influence of different light spectra on chlorogenic acid and rutin contents in callus was analyzed. Callus cultures were incubated in a specially designed plant growth chamber equipped with eight different LED-lights (Nano Bio Light Technology Co., Ltd., Taiwan: http://www.nanobiolight.com/en/). The chamber has two tiers of sections where pertidishes can be kept and exposed to different light spectra by specially designed LED-lids. There were total eight LED-lids (CW-5000 K, WW-2700 K, 8R1B, 7R1G1B, 3R3B3IR, 6R, 6B and 6IR). These LED-lids consisted of single or combinations of four different light spectra emitting blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm) lights. LED-lids CW-5000 K and WW-2700 K represents cool and warm white light while, 5000 K and 2700 K represents color temperature, respectively. Eight LED-lids consisted of the following ratio of light spectra in the order of blue (B): green (G): red (R): infra-red (IR); CW-5000 K (28:43:29:0), WW-2700 K (8:46:46:0), 8R1B (16:0:84:0), 7R1G1B (17:9:74:0), 3R3B3IR (57:0:43:37), 9R (0:0:100:0), 9B (100:0:0:0), 9IR (0:0:0:100) (Personal communication with the Nano Bio Light Technology Co., Ltd., Taiwan). Number (9, 7, 3, 1) in each LED-lid code represents the number of LED chips used for a particular lid. Light intensity among these eight lids varied in the range of 54–64 μmole/m2/s depending upon the type of the LED-lid. Calli were inoculated in petridishes (90 mm) which were kept in horizontal sections in the LED chamber.
Induction of secondary somatic embryos and conversion to plantlets
Vitrified somatic embryos obtained in the ‘LED light experiment’ were cut into tiny pieces forming an embryogenic mass. This mass (200 mg/each petridish) was inoculated on a fresh MS basal medium with ABA (0.5, 1.0, 2.0, 4.0 mg/L), 3 % sucrose and 0.4 % GPP and incubated in a culture room at 25 ± 2 °C in dark. Observations were recorded after 60 days of incubation.
Acclimation and survival of somatic embryos-derived plantlets
For further growth, small plantlets developed in the ‘LED light experiment’ were transferred to glass bottles (650 mL capacity) each containing 100 mL of MS basal medium supplemented with 3 % sucrose and 0.4 % GPP and incubated in a culture room at 25 ± 2 °C, a light and dark cycle of 16/8 h and an illumination intensity of 34 μmol/m2 s. When these plantlets grew about 5–6 cm in length, these were carefully taken out from the bottles, gently washed to remove adhering medium and then transplanted to plastic pots (90 mm dia) containing a mixture of peat soil: perlite: vermiculite (2:1:1). Each pot was covered with a transparent plastic sachet to maintain the humidity. These pots along with sachets were kept in the University greenhouse. After 2 weeks, sachets were removed. Survival rate was recorded after 5 weeks of transplanting.
LC/MS analysis of tissue culture plants and commercially available samples of P. japonicum
Preparation of HPLC/MS standard and samples
Standard samples of chlorogenic acid (ChromaDex) and rutin (Fluka Analytical) were purchased from Sigma-Aldrich Co. LLC. Each standard (10 mg) was taken into a separate 10 mL volumetric flask, and then added methanol to the mark; diluted 10 times into 100 ppm with purified water; took 0.1 mL respectively and further diluted to 10 ppm. The diluted standard solution was filtered with 0.22 μm filter Millipore (Millipore, USA), and then 5 μL was injected for LC/MS analysis. Samples of calli (from the LED light spectra experiment) for the LC/MS analysis were collected from the petridishes and their fresh weights were recorded. All the samples were then freeze-dried for 24 h and their dry weights were recorded. Fraction (100 mg) of each dried sample was crushed into fine powder and dissolved in 10 mL of ethanol. It was ultra-sonicated for 10 min and the supernatant was collected after centrifugation at 5000 rpm for 10 min. This process was repeated three times for each sample. After filtration, ethanol extracts were evaporated to dryness with the help of a rotary evaporator. The residue was dissolved in 10 mL ethanol and filtered through a 0.22 μm (Millipore, USA) membrane before LC–MS analysis.
Instrument and the conditions
The analysis was carried out using a ‘Surveryor HPLC–MS’ pump with an auto sampler. The solution was separated on an Xbridge C18 LC column (2.1 × 150 mm, 5 µm) from Waters (Waters Corp., Milford, MA, USA) at a room temperature. The mobile phases were pure water (Millipore, USA) containing 0.1 % acetic acid (A) and acetonitrile/methanol (9/1, v/v) (B). The gradient was initialized at 80 % A held for 2 min, then increased linearly from 80 % A to 60 % A in 7 min, increased to 10 % A in the following 3 min, further increased to 5 % A in the following 5 min, and then decreased to 80 % A over 1 min. The column was then re-equilibrated at 80 % A for 4 min. The flow rate was 0.2 mL/min. Mass Spectra analyses were performed using a TSQ Quantum ultra EMR tandem Mass Spectrometer (Thermo Electron, San Jose, CA, USA), equipped with an atmospheric pressure ionization (API) interface. The spectra were obtained in positive ESI mode. The spray voltage was 4.5 kV, the capillary temperature was 275 °C, the sheath gas pressure was 35 arbitrary units, and the auxiliary gas was 5 arbitrary units. The mass scan was ranged from m/z 200 to 1000.
Software SAS 9.1 was used for statistical analysis. Data were subjected to the least significant difference (LSD) tested at 5 % probability level (p > 0.05) wherever possible. Each treatment had minimum 20 replicates. The experiments were repeated three times except LC–MS analysis.