Materials and Reagents

Gentian (Gentiana triflora) tissue culture plantlets
Sucrose
Gentiobiose (Sigma-Aldrich, catalog number: G3000-5G )
Gellan gum (Wako USA, catalog number: 075-03075 )
Liquid nitrogen
Milli Q grade water
MS vitamins (see Recipes)
Propagation medium (see Recipes)
IOWB induction medium (see Recipes)
Gentiobiose medium (see Recipes)

Equipment

Sterile magenta boxes (sterilized by autoclaving)
Surgical tape (3M, catalog number: 1530-0 )
Glass culture tubes (sterilized by autoclaving)
Silicon plugs (sterilized by autoclaving)
Sterile 15 ml plastic tubes (such as Greiner Bio-One GmbH, catalog number: 188271 )
Sterile syringe filter (Millipore, catalog number: SLGP033RS )
Sterile petri dishes (IWAKI PUMPS, catalog number: SH90-20 )
Cutoff filter (Millipore, catalog number: UFC5003BK )
Growth chambers
Clean bench
Scalpel
Forceps
Ball miller
Centrifuge
Freeze dryer

Procedure

Preparation and gentiobiose feeding of gentian IOWB and plantlets
1. In vitro shoot cultures of gentian were prepared according to the method of Hosokawa et al. (1996).
2. Transfer shoot tips (approximately 1 cm) to magenta boxes containing 70 ml of propagation medium and culture at 20 °C for 1 to 2 months under a 16/8 h light/dark photoperiod (Figure 1A).
3. Transfer 3 to 5 shoot tips (approximately 1 cm) to a magenta box containing 70 ml of IOWB induction medium (Figure 1B) and culture at 20 °C for over 6 months under a 16/8 h light/dark photoperiod (Figure 1C).
4. Harvest the produced IOWBs and cut into approximately 2 cm length pieces using a scalpel and forceps on a plastic petri dish (Figure 2A).
  
  Figure 1. IOWBs induction from gentian plantlets. A. Cutting shoot tips from plantlet. B. Shoot tips placed on IOWB induction medium. C. IOWBs produced from gentian plantlets cultured for 6 months on IOWB induction medium. Arrows indicate IOWBs. Bar: 2 cm
5. Transfer IOWBs or plantlets to glass culture tubes containing 2 ml of a gentiobiose medium and seal the top of the tubes with silicon plugs (Figure 2B).
  
  Figure 2. Gentiobiose feeding into gentian IOWB and plantlet. A. Harvest of produced IOWB. B. Placement of IOWB and shoot tip on gentiobiose medium. C. IOWB cultured on gentiobiose medium. D. Shoot tip cultured on gentiobiose medium. Bar: 1 cm
6. Culture at 20 °C under a 16/8 h light/dark photoperiod for appropriate period (Figure 2 C-D).
7. Harvest parts of the samples not in contact with the medium.
Detection of gentiobiose in IOWBs or plantlets
1. Freeze-dry and pulverize IOWBs or plantlets in a ball-miller.
2. Homogenize 10 mg dry weight of samples with 500 μl of 50% (v/v) methanol.
3. Centrifuge the homogenates at 20,000 x g for 5 min and filtrate the supernatants through a cutoff filter.
4. Subject the filtrates to thin layer chromatography (Figure 3) or other analyses to detect gentiobiose.
  
  Figure 3. Example analysis of gentiobiose incorporated in gentian plantlets. Extracts from leaves of plantlets cultured on normal MS medium (Control) or MS medium containing 0.05% gentiobiose labeled with rhodamine (Treated) for 3 days were subjected to TLC analysis. STD, 0.01% gentiobiose labeled with rhodamine.

Notes

Propagation and feeding steps should be aseptically performed using a clean bench.
Labeled gentiobiose should be used only for confirmation of incorporated gentiobiose.
For details about gentian IOWBs and plantlets, see Imamura et al. (2014).
For details about TLC analysis, see Takahashi et al. (2014).
For gentiobiose labeling, see the following publications, Hase et al. (1978) and Fry (1997).

Recipes

MS vitamins (100 ml)
10 g myo-inositol
50 mg nicotinic acid
50 mg pyridxine hydrochloride
10 mg thiamine hydrochloride
200 mg glycine
Propagation medium (1L)
4.6 g MS salt
1 ml MS vitamins
30 g sucrose
2 g gellan gum
Adjust pH 5.7 with KOH and autoclave for 15 min
IOWB induction medium (1L)
4.6 g MS salt
1 ml MS vitamins
60 g sucrose
2 g gellan gum
Adjust pH 5.7 with KOH and autoclave for 15 min
Gentiobiose medium (1L)
4.6 g MS salt
1 ml MS vitamins
10 g gentiobiose or labeled gentiobiose^*
2 g gellan gum
Adjust pH 5.7 with KOH and autoclave for 15 min
^*Add to autoclaved MS medium after decrease in temperature to approximately 60 °C
^*Labeled gentiobiose is used for confirmation of incorporated gentiobiose

Acknowledgments

This protocol is adapted from the following publications, Imamura et al. (2014) and Takahashi et al. (2014). This research was supported by a Grant-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, Science and Technology.

References

Fry, S. C. (1997). Novel ‘dot-blot’ assays for glycosyltransferases and glycosylhydrolases: optimization for xyloglucan endotransglycosylase (XET) activity. Plant J 11(5): 1141-1150.
Hase, S., Ikenaka, T. and Matsushima, Y. (1978). Structure analyses of oligosaccharides by tagging of the reducing end sugars with a fluorescent compound. Biochem Biophys Res Commun 85(1): 257-263.
Hosokawa, K., Nakano, M., Oikawa, Y. and Yamamura, S. (1996). Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana. Plant Cell Rep 15(8): 578-581.
Imamura, T., Higuchi, A., Sekine, K. T., Yamashita, T. and Takahashi, H. (2014). High concentration of sucrose induces overwintering bud formation in gentian plantlets cultured in vitro. Plant Biotech 31(2): 97-104.
Takahashi, H., Imamura, T., Konno, N., Takeda, T., Fujita, K., Konishi, T., Nishihara, M. and Uchimiya, H. (2014). The gentio-oligosaccharide gentiobiose functions in the modulation of bud dormancy in the herbaceous perennial gentiana. Plant Cell 26(10): 3949-3963.