In-Vitro Acclimatization of Curcuma Zedoaria Under Iso-Osmotic Treatments, Ex-Vitro Adaptation and Agronomic Traits in Greenhouse Conditions

Low survival rate, poor adaptation to ex-vitro environments, and time required for hardening the plants to cope with uctuated environments of eld trial are identied as major barriers in this technology. In present study, iso-osmotic adjustment in the culture medium using sucrose and/or mannitol was applied to the in-vitro cloning of Curcuma zedoaria (white turmeric) plants, which were transferred to ex-vitro conditions and subsequently cultivated in the greenhouse conditions prior to harvest after 9 months. During both in-vitro and ex-vitro development of plant, growth and physiological traits under 3% sucrose (Suc) + 2.5% mannitol (Man) were lower than those in control (3% Suc; conventional tissue culture). Interestingly, pseudostem height and root length in acclimatized plantlets under 3% Suc + 2.5% Man were sharply dropped by 60.13% and 92.37% over control, respectively, resulting in a decrease in the ex-vitro adaptation by 56.27% and 33.33% over the control. A positive relationship between reduction of net photosynthetic rate (P n ) and sucrose concentration in the leaf tissues was evidently observed. Remarkably, the morphological and physiological traits of aboveground and underground parameters of acclimatized plantlets under 3% Suc + 2.5% Man were maximized over control, leading to high yield of curcuminoids (229.4 mg plant − 1 ) in the dry rhizome (31 g plant − 1 ) when cultivated under greenhouse microenvironments for 9 months. Based on this investigation, we propose that plantlets of C. zedoaria micropropagated using 3% Suc + 2.5% Man can readily acclimatize under ex-vitro conditions and subsequently develop as healthy plants with compact and uniform size. and ex-vitro adaptation and agronomic traits under greenhouse conditions. leaf


Introduction
Curcuma zedoaria (Christm.) Roscoe (white turmeric; Zingiberaceae) is a perennial rhizomatous plant, commonly used as a food ingredient and traditional medicine. It is originated in Bangladesh, India and Sri Lanka and distributed throughout Asia including China, Japan, Nepal, Thailand and Vietnam (Lobo et al. 2009). Essential oil in several parts of the plant including mature rhizome, roots, pseudostem shoots and leaves have been used in ethnomedicinal practices (Ayati et al. 2019). Likewise, its leaf extracts are used for dropsy and leprosy, fresh roots cure leucorrhoeal discharge and powdered rhizome is antiallergic in nature (Lobo et al. 2009). Rhizome of C. zedoaria contains curcuminoids, which are speci c to Curcuma species and widely used in pharmacology for anti-cancerous, antiin ammatory and antimalarial treatments (Paramapojn and Gritsanapan 2009;Peng et al. 2010;Jena et al. 2020). Demethoxycurcumin (DEM) has been identi ed as the major compound in the rhizome of C. zedoaria along with curcumin (CUR) and bisdemethoxycurcumin (BIS) as the minor compounds (Syu et al. 1998; Burapan et al. 2020). Moreover, curcuminoid biosynthesis related genes, i.e., diketide-CoA synthase (DCS), curcumin synthase 1 (CURS1), curcumin synthase 2 (CURS2), and curcumin synthase 3 (CURS3) are identi ed and characterized as major routes for their production in the rhizome of white turmeric (Lan et al. 2018). Seasonal variations are one of the most important environmental factors, which regulate uctuations in the major compounds including curcumenol and dihydrocurdione (Pamplona et al. 2005). Mother rhizome propagation has been wildly practiced for cultivation of white turmeric but it results in slow growth rate, non-uniformity in the produce and the risk of soil-borne infections, which are major concerns in its production at commercial scale (Ajitomi et al. 2015; Prameela and Bhai 2020). Micropropagation in elite clones of C.zedoaria has been well established to overcome the barriers for a large-scale production ( Plant micropropagation via in-vitro culture under aseptic conditions is a novel technology to produce elite clones as mother stock so as to facilitate the industrial scale production. Acclimatization of the in-vitro plantlets before transferring them to eld conditions is a critical concern since sudden change from a delicate growth conditions with high nutrients, high relative humidity and low light intensity to harsh environmental conditions with resource competition, uctuating climate and uneven light intensity (Hazarika 2006;Kumar and Rao 2012). Several factors affect the success of this process of hardening, i.e., CO 2 concentration, relative humidity, suitable substrates, inoculated arbuscular mycorrhizal fungi and optimized carbon sources in the media ( Maiti and Pramanik 2013). Limited information is available in the literature regarding effect of the combined application of sucrose (carbon source) and mannitol (osmotic adjustment) in the culture medium for in-vitro hardening of plantlets. The aim of this study was to investigate the impact of iso-osmotic conditions of C. zedoaria on in-vitro acclimatization and exvitro adaptation and agronomic traits under greenhouse conditions.

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 (Clorox â , 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 ux (PPF) intensity provided by uorescent 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.

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 Osmocote â 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 lter 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 uorescence emission including maximum uorescence (F v /F m ) and photon yield of PSII (F PSII ) from the adaxial surface of second fully expanded leaf of the shoot tip was measured using a uorescence monitoring system (model FMS 2; Hansatech Instruments Ltd., Norfolk, UK) (Loggini et al. 1999;Maxwell and Johnson 2000).

Biochemical assays
Curcuminoids assay, the dried rhizomes were ground into ne 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 ltered (Whatman â #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 ltrated through 0.45 µm (Millipore TM , Nihon Millipore Ltd., Japan) nylon lter. 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 C 18 (VertisepÔ UPS C 18 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 ow 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 Window Ò , SPSS Inc., Chicago, USA). Relationships between physiological and morphological data of each treatment were validated using Pearson's correlation coe cient.
Microrhizome fresh and dry weights in plantlets acclimatized under 3% Suc (control, -1.0 MPa) were increased to 0.44g and 35.7 mg, respectively, when compared with iso-osmotic treatments ( Fig. 2E and 2F). A positive relation between decreasing root osmotic potential and root fresh weight (
In the present study, plantlets acclimatized under 4% Man were undeveloped and consequently died when transplanted in the peat moss substrate. On the other hand, the plantlets subjected to iso-osmotic treatments (6% Suc and 3% Suc + 2.5% Man; -1. There are so many steps to acclimatize the plants in greenhouse conditions including ex-vitro hardening, evaporation prevention, mist spray to keep high relative humidity, and reduced transpiration rate from plantlets, etc. A novel protocol to harden plantlets is required to facilitate their direct transplantation to ex-vitro environments. Previously, survival percentage of carob tree (Cerafoma siliqua) was signi cantly improved by adding 58 mM glucose + 58 mM mannitol in the culture medium (Custódio et al. 2004). Similarly, shoot and root traits of bromeliad plants (Vriesea in ata) derived from in-vitro sucrose containing medium were decreased (Freitas et al. 2015), in relation to concentrations (4-6% Suc) and plant species (Wojtania et al. 2019). In addition, root traits in the plantlets of magnolia cv. 'Yellow Bird' (cross between Magnolia acuminata and M. × brooklynensis) acclimatized under 3-4% Suc were inhibited, resulting in more than 75% of leaf necrosis in the plants (Wojtania et al. 2019). P n of adapted plantlets derived from in-vitro acclimatization under 3% Suc + 2.5% Man was signi cantly declined by 22.81% over control (Fig. 5E), leading to decreased sucrose concentration in the leaf tissues (Fig 6A; R 2 = 0.5636). Sucrose concentration in the leaf tissues of in-vitro acclimatized plants under iso-osmotic potential (-1.3 MPa) was dropped by 43.65-47.09% over control (Fig. 5F). Therefore, glucose, fructose and total soluble sugar in the leaf tissues were unaffected. Physiological parameters such as leaf greenness (SPAD), F v /F m , F PSII , E and g s in the second fully expanded leaves of each treatment were found to be unchanged (Table 3). In addition, strong relationships between leaf area and microrhizome dry weight ( Fig. 6B; R 2 = 0.9478), pseudostem height and root length ( Fig. 6C; R 2 = 0.7555), and number of leaves and leaf area ( Fig. 6D; R 2 = 0.8396) were noticed.
Physiological strategies of plants derived from in-vitro acclimatization, such as water loss prevention by stomata and CO 2 assimilation to produce large amount of carbohydrate for plant growth and development have been validated in relation to eld conditions (Seon et al. 2000). In general, maintained P n along with limited transpiration (E) by stomata is a good indicator for rapid ex-vitro adaptation of

Long-term cultivation of white turmeric and agronomic traits
Morphological characteristics of white turmeric plants observed during its entire life cycle from ex-vitro adaptation to nal harvest are presented for all the treatments in Fig. 7. Aboveground traits, i.e., pseudostem height, number of leaves, leaf length and leaf width in all the treated plants were unaffected. It means that there is uniformity in the aboveground parameters of white turmeric plants irrespective of their in-vitro treatments. Remarkably, pseudostem fresh-and dry-weights were maximal in plants derived from 3% Suc + 2.5% Man acclimatization (1.53 and 1.31-folds over control, respectively) ( Fig. 8A and 8B). Interestingly, the underground parameters including root length, number of roots, rhizome length and rhizome width of plants derived from 3% Suc + 2.5% Man treatment were signi cantly improved by 3.54, 1.5, 1.69 and 2.71-folds over control, respectively (Table 4). Root fresh weight, root dry weight, rhizome fresh weight and rhizome dry weight of plants derived from 3% Suc + 2.5% Man treatment were strongly enhanced upon -1.3 MPa iso-osmotic treatments and recorded to be 145.93 g (Fig. 8C), 10.7 g (Fig. 8D), 248.39 g (Fig. 8E) and 31.0 g (Fig. 8F), respectively. In white turmeric, DEM in the rhizome was identi ed as major compound, whereas BIS and CUR were the minor compounds ( Fig. 9A-9C). The BIS, DEM, CUR, total curcuminoids and curcumin yield in plants derived from 3% Suc + 2.5% Man treatment were peaked at 0.7 mg, 5.9 mg, 0.8 mg, 7.4 mg and 229.4 mg plant -1 , respectively (Fig. 9A-9E). A positive relation between rhizome dry weight and total curcuminoids were observed ( Fig. 9F; R 2 = 0.9467). In term of plant physiological responses, leaf greenness (SPAD) and P n in plants derived from 3% Suc + 2.5% Man treatment were increased by 1.34 and 1.22-folds, respectively (Table 5). In addition, E and g s in acclimatized plants under 6% Suc and 3% Suc + 2.5% were signi cantly improved over controlled plants (Table 5). In contrast, F v /F m and F PSII in plants were unaffected irrespective of the treatment.
After 9 months, overall growth performances in both aboveground and underground traits of in-vitro plantlets acclimatized under 3% Suc + 2.5% Man were better than those in control, including total curcuminoids in the mature rhizome. Overall growth performances and yield traits of plants derived from Man osmopriming were signi cantly better than non-priming control (Kaur et al. 2005; Papastylianou and Karamanos 2012). In C. zedoaria, the morphological parameters, i.e., plant height, number of tillers, number of leaves, leaf length, leaf width, rhizome diameter, total rhizome weight and rhizome length as well as secondary metabolites in rhizome and leaf oil were nearly same in mother plant and in-vitro derived plants under eld trial (Jena et al. 2020). In tea (Camelia sinensis), the major compounds, epigallocatechin and epigallocatechin gallate, in the fresh leaves of plantlets acclimated under 200 mM mannitol were enriched over control (Samarina et al. 2020). Fresh mass, dry mass, number of shoots and COX1-inhibition activity (%) in plants of Eucomis autumnalis acclimatized under 4% Suc were increased when compared with low sucrose treatment (2%; w/v) (Taylor and van Staden 2001). It was con rmed that the physiological and morphological traits as well as curcuminoids yield of in-vitro plantlets acclimatized using iso-osmotic treatment of 3% Suc + 2.5% Man were better compared to control during long-term cultivation. In Ruta graveolens, plant morphological characters in mannitol acclimatized plantlets were unaffected, whereas coumarins and rutin in terms of content and total yield were regulated in response to increase in Man concentration in the culture medium (Mohamed and Ibrahim 2012).

Conclusions
The present study demonstrated a successful hardening of in-vitro plantlets of white turmeric using 3% Suc + 2.5% Man (-1.3 MPa) resulting in compaction by ∼50% in terms of aboveground and underground growth performances, thereby requiring small area for master stock production. Healthy plants (uniform-sized) derived from in-vitro acclimatization under 6% Suc and 3% Suc + 2.5% Man (-1.3 MPa) treatments were rapidly adapted to ex-vitro greenhouse conditions within 6 weeks, and subsequently adapted to long-term greenhouse microclimates. Interestingly, physiological characters including photosynthetic abilities and underground rhizome traits in plants derived from 3% Suc + 2.5% Man in-vitro acclimatization were improved, leading to an increase in rhizome and curcuminoids yield. Tables   Table 1 Pseudostem Table 4 Pseudostem height (Ps-H), number of leaves (NL), leaf area (LA), leaf length (LL), leaf width (LW), root length (RL), number of roots (NR), rhizome length (RhL) and rhizome width (RhW) of C. zedoaria plants derived from in vitro iso-osmotic treatments, subsequently transplanted to soil substrate under greenhouse conditions for 9 months. Data represented as mean ± standard error (SE) (n = 4).