In-vitro acclimatization
Mother rhizomes of C. zedoaria were collected from Nakhon Nayok province, central region of Thailand (latitude 14.090956, longitude 100.932454) and the rhizomes were allowed to sprout to give rise to new shoot buds. The sprouted shoots with 1 cm length were dissected and surface-sterilized with 1% hypochlorite solution (Cloroxâ, 6% sodium hypochlorite. v/v; ai, Clorox Company, Oakland, USA) for 15 min and washed thrice using 100 mL sterile distilled water. Leaf sheaths were removed, and apical shoot was inoculated onto 35 mL Murashige and Skook (MS) medium containing 3% (w/v) sucrose and 1 mg L-1 BA in the 125 mL glass vessel. Then, culture vessels containing plantlets were incubated under 60±5% relative humidity (RH), 25±2°C ambient temperature, and 60±5 μmol m−2 s−1 photosynthetic photon flux (PPF) intensity provided by fluorescent lamps (Cool white, Philips, Thailand) with a 16 h d−1 photoperiod for 30 days. Single shoots were dissected and transferred to the fresh MS medium on monthly basis for shoot proliferation. The single plantlet (2.0±0.2 cm in length) was transferred to MS medium containing 3% sucrose (control; ‒1.0 MPa) and iso-osmotic conditions at ‒1.3 MPa using 6% sucrose (Suc), 4% mannitol (Man) and 3% sucrose + 2.5% mannitol with 1 plantlet per glass vessel. After 45 days of in-vitro acclimatization, pseudostem height, number of leaves, leaf area, pseudostem fresh and dry weight, leaf osmotic potential, root length, number of roots, root fresh and dry weight, and root osmotic potential of acclimatized plantlets under isosmotic conditions were collected.
Ex-vitro adaptation
Plantlets derived from in-vitro acclimatization using isosmotic conditions were directly transplanted to peat moss substrate (Kâ1913 Class man, K-Select Aquasave, Germany), irrigated regularly and incubated in a greenhouse under 28 ± 2 °C ambient temperature, 500–1000 μmol m-2 s -1 photosynthetic photon flux density with a 10 h d-1 photoperiod, and 80 ± 5% relative humidity for 2 weeks, subsequently transferred to plastic bag (5´14 cm) containing 1 kg garden soil (EC = 2.687 dS m-1; pH = 5.5; organic matter = 10.36%; total nitrogen = 0.17%; total phosphorus = 0.07%; total potassium = 1.19%) and daily irrigated in a greenhouse condition for 4 weeks. Thereafter, pseudostem height, number of leaves, leaf area, root length, number of roots, pseudostem fresh and dry weight, root fresh and dry weight, photosynthetic abilities, and soluble sugars in leaf tissues were measured.
Long-term cultivation and agronomic traits
Ex-vitro adapted plants in each treatment were subsequently transferred to plastic bags (14´30 cm) containing 5 kg garden soil in a greenhouse for 9 months. Daily irrigation and 10 g Osmocoteâ 13-13-13 slow-release fertilizer were supplied as per water and fertilizer application schedule. Pseudostem height, number of leaves, leaf length, leaf width, pseudostem fresh and dry weight, root length, number of roots, root fresh and dry weight, rhizome length, rhizome width, leaf greenness, maximum quantum yield of PSII, photon yield of PSII, net photosynthetic rate, transpiration rate, and stomatal conductance were measured after harvest. In addition, bisdemethoxycurcumin (BIS), dimethoxycurcumin (DEM), curcumin (CUR) and total curcuminoids in the rhizomes were also measured.
Morphological characteristics
Pseudostem height, number of leaves, leaf length, leaf width, leaf area, pseudostem fresh and dry weight, root length, number of roots, root fresh and dry weight, rhizome length, rhizome width, rhizome fresh and dry weight in white turmeric plants were measured as growth parameters. Leaf area was measured by Leaf Area Meter (Model CL-203, CID® Inc, WA, USA). Pseudostem, rhizome and root of white turmeric plants were dried at 80°C in a hot air oven for 48 h, and then placed in a desiccator before the measurement of dry weight.
Physiological characteristics
Osmotic potential in the root and leaf tissues of each treatment was measured according to Lanfermeijer et al. (1991). In brief, 10 mL cell sap was dropped directly onto a filter paper in an Osmometer chamber (5520 Vapro®, Wescor, Utah, USA). Then, the osmolarity (mmol kg−1) was converted to osmotic potential (MPa) using conversion factor of osmotic potential measurement according to Fu et al. (2010).
Leaf greenness (SPAD value) in the second fully expanded leaf from the shoot tip of each treatment was measured using Chlorophyll meter (Model SPAD-520Plus, Konica Minolta, Osaka, Japan) according to Hossain et al. (2015).
Chlorophyll fluorescence emission including maximum fluorescence (Fv/Fm) and photon yield of PSII (FPSII) from the adaxial surface of second fully expanded leaf of the shoot tip was measured using a fluorescence monitoring system (model FMS 2; Hansatech Instruments Ltd., Norfolk, UK) (Loggini et al. 1999; Maxwell and Johnson 2000).
Net photosynthetic rate (Pn; mmol m-2 s-1), stomatal conductance (gs; mmol CO2 m-2 s-1), intracellular CO2 concentration (Ci; mmol mol-1) and transpiration rate (E; mmol m-2 s-1) of second fully expanded leaf of the shoot tip were measured by a portable photosynthesis system (LI 6400XT, LI-COR, Lincoln, NE, USA), following the method of Cha-um et al. (2007).
Biochemical assays
Curcuminoids assay, the dried rhizomes were ground into fine powder in the mortar with liquid nitrogen. Twenty milligrams of the powder was then taken into a glass vial and 5 mL of methanol was added. The mixture was mixed thoroughly by vortex followed by sonication for 30 min. Then, the solution was filtered (Whatmanâ #1, Maidstone, UK) and crude extract was dried by allowing the methanol to evaporate. Curcuminoid content in crude extract was analyzed using High Performance Liquid Chromatography (HPLC). Curcuminoids were dissolved in 1 mL methanol (HPLC grade) and then filtrated through 0.45 µm (MilliporeTM, Nihon Millipore Ltd., Japan) nylon filter. Ten microliters of sample were injected into injection loop and analyzed by HPLC (Waters Associates, Milford, MA, USA) equipped with photodiode array detector (Water 2998) at 425 nm. Bis-demethoxycurcumin (BIS), demethoxycurcumin (DEM) and curcumin (CUR) were separated using C18 (VertisepÔ UPS C18 HPLC) column under 25°C room temperature. The mobile phase consisted of acetonitrile (100% HPLC grade) and acetic acid (0.25%, v/v). The elution was carried out with a gradient set at a flow rate of 0.8 mL min-1. The solvent gradient was 50% acetonitrile for 0 to 8 min, then 50% to 40% acetonitrile from 8 to 10 min, 40% acetonitrile constant from 10 to 15 min, and 40 to 50% acetonitrile from 15 to 16 min (Pothitirat and Gritsanapan 2007).
Sucrose, glucose, and fructose in the leaf tissues of second fully expanded leaf from the shoot tip) were extracted using nanopure water and then the contents of soluble sugar were assayed by HPLC according to the method of Karkacier et al. (2003).
Experimental layout and statistical analysis
The experiment was arranged as Completely Randomized Design (CRD) with four replicates (n=4). The mean values obtained from all treatments were compared using Tukey’s HSD test and analyzed by SPSS (Statistical Package for Social Science) software (version 11.5 for WindowÒ, SPSS Inc., Chicago, USA). Relationships between physiological and morphological data of each treatment were validated using Pearson’s correlation coefficient.