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In vitro grown thickened taproots, a new type of soil transplanting source in Panax ginseng

Background The low survival rate of in vitro regenerated Panax ginseng plantlets after transfer to soil is the main obstacle for their successful micropropagation and molecular breeding. In most cases, young plantlets converted from somatic embryos are transferred to soil. Methods In vitro thickened taproots, which were produced after prolonged culture of ginseng plantlets, were transferred to soil. Results Taproot thickening of plantlets occurred near hypocotyl and primary roots. Elevated concentration of sucrose in the medium stimulated the root thickening of plantlets. Senescence of shoots occurred following the prolonged culture of plantlets. Once the leaves of plantlets senesced, the buds on taproots developed a dormant tendency. Gibberellic acid treatment was required for dormancy breaking of the buds. Analysis of endogenous abscisic acid revealed that the content of abscisic acid in taproots with senescent shoots was comparatively higher than that of taproots with green shoots. Thickened taproots were transferred to soil, followed by exposure to gibberellic acid or a cold temperature of 2°C for 4 mo. Cold treatment of roots at 2°C for 4 mo resulted in bud sprouting in 84% of roots. Spraying of 100 mg/L gibberellic acid also induced the bud sprouting in 81% roots. Conclusion Soil transfer of dormant taproots of P. ginseng has advantages since they do not require an acclimatization procedure, humidity control of plants, and photoautotrophic growth, and a high soil survival rate was attained.


Introduction
Korean ginseng plants (Panax ginseng Meyer) are herbaceous medicinal plants that grow slowly. A cultivation period of > 4 yr is required before the plants set seeds. Therefore, plant tissue culture is an alternative method for the clonal propagation and breeding of ginseng. Plant regeneration via somatic embryogenesis in P. ginseng has been reported [1e6]. However, there are a few reports of successful field transfer of tissue culture-raised ginseng plantlets [3,7]. Serious problems are encountered during acclimatization and soil/field transfer of plantlets. The in vitro grown plantlets are feeble and can be affected by fungal infections [3,7]. P. ginseng is a perennial plant with storage taproots. The leaves and stems die at the end of the growing season. The storage taproots survive under the ground during winter, and new shoots develop from overwintering buds on taproots. Small ginseng taproots are mainly used as planting stocks for continuous cultivation in open fields.
We observed the in vitro taproot thickening of plantlets after prolonged culture. In this study, we investigated the effect of sucrose on the taproot thickening of in vitro grown plantlets, in addition to the effects of gibberellic acid (GA 3 ) and cold temperatures on the bud dormancy breaking of taproots. Soil transfer of in vitro grown thickened taproots of ginseng plantlets was successfully accomplished.

Germination and plant regeneration
Stratified seeds of Korean ginseng were harvested from a research field of the Department of Herbal Crop Research, National Institute of Horticultural and Herbal Science (Chungcheongbuk-do, Korea), RDA. Embryogenic callus and somatic embryogenesis were established as described earlier [7]. Germination of somatic embryos was induced by transferring embryos onto 1/2 Schenk and Hildebrandt (SH) medium with 5 mg/L GA 3 . Plantlets were maintained on 1/2 strength SH [8] medium with 3% sucrose for several months without subculture. During this prolonged culture period, leaf senescence and thickening of roots were monitored, and photographs were taken if necessary. To investigate the role of GA 3 on the bud growth on taproots, defoliated taproots were transferred onto 1/2 SH medium with different concentrations (0 mg/L, 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L) of GA 3 . After 1 mo of culture, the growth of shoots and roots was assessed. Four plants were cultured in polycarbonate Magenta culture vessels (Sigma Chemical Co., St Louis, MO, USA). Twenty explants were used per treatment, and each treatment was repeated three times. Plantlets were cultured in polycarbonate Magenta culture box or 300 mL glass bottles.

Effect of sucrose on the growth of taproots
Somatic embryo-derived plantlets with roots and shoots (approximately 10 mm in length) were transferred onto 1/2 SH medium with various concentrations of sucrose (0%, 1%, 2%, 3%, 4%, and 5%) in a 500 mL recipient for automated temporary immersion (RITA) bioreactor (Cirad Biotrop, Montpellier Cedex, France). After 2 mo of culturing, the growth of roots and shoots was monitored by measuring the lengths of shoots and roots and the diameters of roots. Twenty explants were cultured in the bioreactor, and each treatment was repeated three times.

Soil transfer of thickened taproots after cold and GA 3 treatment
Taproots with dormant buds (3 mm diameter of roots) were transferred to soil in plastic square boxes (25 Â 20 Â 15 cm 3 ) containing perlite and peat (3:1 v/v), and then treated with 100 mg/ L GA 3 or exposed to a cold temperature of 2 C or 10 C for 4 mo. The rate of sprouting was monitored after transfer of the soil boxes to a culture room maintained at 24 C. Fifteen roots were planted in a box. Thirty roots were used per treatment, and each treatment was repeated three times.

Abscisic acid analysis
Plantlets at various stages of development (cotyledonary somatic embryos, germinated embryos, taproots with green shoots, and taproots with senescent shoots) were sampled for the analysis of endogenous abscisic acid (ABA) content. Samples were ground in a mortar with liquid nitrogen, and the powdered samples were transferred into a tube and mixed with 80% methanol with 1% acetic acid and butylated hydroxytoluene. After vortexing of liquids for 1 min and sonication for 10 min at 0 o C, the sample mixtures were centrifuged at 19,750 g, 4 C, for 10 min. The supernatants were collected, and the pellets were re-extracted with 3 mL of the extraction solvent. The second group of extracts was centrifuged, and then supernatants were combined and dried completely in a rotavapor.
The extracts were reconstituted in 200 mL of acetonitrile/water/ acetic acid (90:10:0.05, v/v), stirred, vortexed, centrifuged (13,167 g, 10 min), and filtered through a 0.45 mm PTFE filter (Waters, Milford, MA, USA), and 10 mL of the extracts were injected into an liquid chromatography-ion trap-time of flight-mass spectrometer (LC-IT-TOF-MS) system (Shimadzu, Kyoto, Japan) equipped with an ESI source used in positive and negative ion modes. Quantification was performed using the standard addition method by spiking control plant samples with ABA solutions (ranging from 10 ng/mL to 200 ng/mL).
LC experiments were conducted on a Shimadzu (Kyoto, Japan) HPLC system that consisted of an LC-20AD binary pump, a DGU-20A degasser, an SIL-20A autosampler, a CTO-20AC column oven, and an SPD-M20A PDA detector. The mobile phase (delivered at 0.5 mL/min) comprised solvent A (H 2 O) and solvent B (CH 3 CN). A binary gradient elution was performed for ABA analysis: initial 25% B for 1.0 min, linear gradient 25e35% B from 2 min to 5 min, linear gradient 35e55% B from 6 min to 15 min, linear gradient 55e70% B from 16 min to 20 min, and a final quick return to the initial 25% B, which was maintained for 10 min to balance the column. Chromatographic separation was achieved on an Eclipse XDB-C18 column (5 mm, 3.0 Â 250 mm 2 ; Agilent, Santa Clara, USA) at 40 C.
The LC-IT-TOF-MS mass spectrometer (Shimadzu) equipped with an ESI source was used in the negative ion mode. The following optimized analytical conditions were used: detector voltage, 1.60 kV; nebulizing gas (N 2 ) flow, 1.5 L/min; dry gas (N 2 ) flow, 50 kPa; pressure of TOF region, 1.5 Â 10 À4 Pa; ion trap pressure, 1.7 Â 10 À2 Pa; and ion accumulation time, 30 ms. For the qualitative analysis, the scan ranges were set at m/z 100e500 for MS 1 ; ultra-high-purity argon was used for cooling. Standard ABA was directly examined under the same conditions.

Results and discussion
3.1. In vitro taproot thickening of plantlets converted from somatic embryos Somatic embryos germinated rapidly and converted into plantlets on transfer to the medium supplemented with more than 5 mg/L GA 3 (Fig. 1A). Germinating somatic embryos were transferred onto 1/2 SH medium with 3% sucrose. Rapid growth of shoots and roots was achieved within 2 wk of culture (Figs. 1B, 1C). Following elongation of the plantlets, thickening of taproots was observed in the hypocotyl and primary root regions (Fig. 1D). The plantlets exhibited obviously thickened taproots with welldeveloped shoots (Figs. 1E, 1F). In plants with fleshy taproots, such as ginseng, carrot, radish, and beet, primary roots with hypocotyls become enlarged and function as storage taproot organs [9]. We found that in vitro cultured ginseng plantlets exhibited the same pattern of root thickening as that observed in taproot formation in the other plants.

Effect of sucrose on the growth of plantlets and root thickening
Small plantlets, just after germination of somatic embryos, were transferred into an RITA temporary immersion bioreactor ( Fig. 2A) containing various concentrations (1%, 2%, 3%, 4%, and 5%) of sucrose. Fresh weights of shoots and roots were affected by the sucrose concentration. At a high sucrose concentration (5%), the fresh weight of roots was promoted but that of shoots (Figs. 2Be2D) was reduced. Accordingly, the growth of plantlets and root thickening were highly affected by the sugar concentration in the medium. As the sucrose concentration increased, the length of shoots and roots of plantlets were reduced (Fig. 3A), but root thickening was conspicuously enhanced (Fig. 3B). These results suggest that an excess amount of sucrose absorbed by plantlets may stimulate the secondary growth of roots. In Lilium, a higher concentration of sucrose has been shown to increase the size of bulb scales. The combination of sucrose and mannose has also been demonstrated to promote bulb growth [10]. In shoot cultures of yam (Dioscorea composita), 8% sucrose was found to be the most significant medium constituent for promoting microtuber induction [11].  shoot parts (leaves) of plantlets turned yellow after prolonged culture (more than 3 mo), indicative of the senescence of leaves. Thickened taproots with tiny buds were obtained after leaf senescence (Fig. 4A). All the taproots had obvious shoot buds on their heads (Fig. 4B). Although the taproots with buds were subcultured on a new hormone-free medium, growth of shoots from buds did not occur (Fig. 4C). This result indicates that bud dormancy of taproots also occurred when the leaves died following prolonged culturing. GA 3 was a prerequisite for the bud sprouting of these taproots. The optimal GA 3 concentration for the sprouting of buds, which is a suitable concentration for the breaking of buds, was 10 mg/L (Figs. 4D, 4E). Owing to their dormancy, taproots can be preserved for > 6 mo. All the dormant taproots regenerated into plantlets after the GA 3 treatment. Leaf senescence in plants grown in nature can occur prematurely under unfavorable environmental conditions [12]. The primary reason for leaf senescence in P. ginseng plantlets might be the deprivation of nutrition in the medium because, in this study, senescence occurred only after prolonged culture. Senescence and dormancy of plantlets caused by deficiency of nutrients have been reported in bulbous plant species when they are not subcultured [13].

Dormant taproot formation after senescence of plantlets
Various phytohormones, including ABA, salicylic acid (SA), jasmonic acid (JA), and ethylene, are associated with leaf senescence. ABA is considered a plant stress hormone. It has been well known that ABA treatment stimulates leaf senescence [14]. Dormancy of plantlets occurs frequently during in vitro culture [15]. Leaf senescence and dormancy of buds on thickened ginseng roots might be attributed to endogenous ABA accumulation in plantlets after prolonged culture.
Endogenous ABA content was highest in taproots with senescent leaves (78.5 ng/g FW) compared with those with green shoots (34.2 ng/g FW) (Fig. 5). ABA content in taproots with senescent leaves was remarkably reduced (38.4 ng/g FW) after 4 mo of cold treatment at 2 C (Fig. 5). Germinating somatic embryos induced by GA 3 treatment had the lowest content of ABA (12.7 ng/g FW). This result revealed that the dormancy of buds of taproots with senescent leaves might be attributed to the relatively high content of endogenous ABA.

Soil transfer of in vitro thickened taproots
After the removal of aged leaves, all thickened taproots with healthy dormant buds (Fig. 6A) were transferred to soil, and were then exposed to a cold temperature of 2 C or 10 C for different durations or treated with GA 3 . The taproots did not produce any shoots unless they were exposed to either GA 3 or a cold temperature (Fig. 6B). When pots containing taproots were exposed to a cold temperature (2 C) for 4 mo and then returned to room temperature, bud sprouting occurred on 84% of taproots (Figs. 6C, 6D, 6F). Cold treatment at 10 C was not effective for the dormancy breaking of buds because only 37% of roots exhibited sprouting after 4 mo of this treatment. Spraying of water containing 100 mg/L GA 3 also effectively induced the sprouting of buds on 81% of taproots (Fig. 6E). Spraying of GA 3 at a concentration of 20 mg/L or 40 mg/L was not effective because the sprouting of buds did not exceed 50% (data not shown). There are some differences in the number of sprouting leaflets between the cold and GA 3 treatment. Only a single leaflet developed on the taproots after cold treatment (Figs. 6C, 6D), but two leaflets were common after GA 3 treatment (Fig. 6E).
Choi et al. [3] described the transfer of actively growing in vitro ginseng plantlets to soil. Only 27% of the plantlets survived for up to 15 wk after transfer to a greenhouse [3]. The soil survival rate achieved by the soil transfer of in vitro thickened taproots was markedly increased compared with that observed with the transfer of young plantlets [3]. Dormant taproots can be preserved for a long period of time in vitro, and they can very easily be transferred to soil.
During in vitro culture, plantlets grow under very special conditions in relatively airtight cultivation vessels (high humidity) and a carbon-rich condition, mainly in the sucrose medium as an energy source. After ex vitro transfer, plantlets might easily be impaired by sudden changes under low humidity and photoautotrophic condition, therefore transplantation of plantlets usually needs some period of acclimatization with gradual lowering in air humidity [16]. In the soil transfer of thickened taproots of ginseng, special treatment for humidity control is not required for ex vitro acclimatization because shoot sprouting occurs after ex vitro transfer. Thus, the soil transfer of in vitro thickened taproots may have several advantages for increasing the soil survival of in vitro propagated P. ginseng plantlets. There are 12 recognized ginseng species in the world depending on the method of classification [17]. This protocol can also be adopted for soil transfer of micropropagated plants of other ginseng species.