| Plant latin name | Gentiana triflora Pallas |
| Literature code | Gentiana-triflora-Ref-3 |
| Reference | Hosokawa K et al., Plant Cell Reports 15: 578-581 (1996) |
| Summary | Several culture conditions were examined for promoting efficient plant regeneration from explants of Gentiana. Adventitious shoot regeneration from leaf explants of cv.WSP-3 was very superior on MS medium, compared to B5medium, supplemented with four cytokinins (TDZ, 4PU-30,BA and zeatin). An auxin / cytokinin combination was required for regeneration. TDZ was the most effective cytokinin, while NAA was more effective than IAA or 2,4-D.Optimum conditions for regeneration from explants (leaf,stem and root) of cv. WSP-3, evaluated in terms of regeneration frequency and number of regenerated shoots per explant, were TDZ and NAA in combination, 5 - 10 mg/1 and 0.1 mg/l for leaf and stem explants, and 10 mg/l and 1 mg/l for root explants, respectively. Application of these conditions to eight other commercial cultivars resulted in 30-100% regeneration from leaf explants. The number of chromosomes in each of ten regenerated plants of each cultivar was diploid, 2n=26. Shoots regenerated in vitro were rooted in phytohormone-free medium and transferred to soil. |
| Objectives | Adventitious shoot regeneration from leaf, stem and root explants |
| Materials | Seeds of five Gentiana triflora cultivars (F1 hybrid) Homoi, Ihatovo, Iwate, lwate-otome and Maciry; an interspecific hybrid cultivar (F1 hybrid, G. triflora x G. scabra ) Albireo; and three in vitro shoot cultures of interspecific hybrid cultivars (clonal variety, G. triflora x G. scabra) H-3, Polarno white and WSP-3, were obtained from Iwate Horticullural Experiment Station. The in vitro shoot culture of cv. WSP-3 was used mainly to establish optimal conditions for adventitious shoot regeneration |
| Explant | Seeds, leaf (approximately 5 mm square), stem and root explants (approximately 5 mm in length) from in vitro cultures from the seedling or shoot cultures |
| Initial culture | Seeds were surface-disinfected with 1% sodium hypochlorite solution for 5 min followed by two rinsings in sterilized distilled water. The disinfected seeds were germinated on MS medium supplemented with 30 g/l sucrose and 2 g/l gellan gum. All axillary buds were cultured on the same medium. Plantlets from the seedlings or in vitro shoot cultures were routinely subcultured at 20℃ for 16 h daytime under fluorescent lamps (50 µmol m-2 s-l). Subcultures were made once every two months by transferring terminal and lateral cuttings into the same medium. |
| Shoot multiplication | Adventitious shoot regeneration from leaf explants of cv.WSP-3 was more frequent on MS medium than B5 medium in all cases. TDZ (1-10 mg/L) in combination with NAA (0.1 mg/L) proved to be optimal for regeneration, followed by 4PU-30, BA and zeatin. The presence of an auxin was required, as no regeneration was obtained with cytokinin alone.
In order to establish optimum conditions for regeneration, TDZ was used in combination with three auxins. NAA was the most effective for regeneration from leaf explants at 0.1 mg/L when combined with 5-10 mg/L TDZ (regeneration frequency, 100%). Although 100% regeneration frequency was also attained on MS medium containing 20 mg/L TDZ along with 0.1 mg/L NAA and 1 mg/L
IAA, some of the shoots were vitreous, which hindered subsequent rooting and acclimation. 2,4-D was the least suitable.
Regeneration from stem and root explants was greatest at 5-10 mg/L TDZ in combination with 0.1 mg/L NAA, and at 10 mg/L TDZ in combination with 1 mg/L NAA, respectively. For root explants, only five adventitious shoots per explant were formed which is less than under the optimum conditions for leaf and stem explants. Leaf and stem explants were found to be most suitable for shoot regeneration.
Adventitious shoot regeneration from the leaf explants of nine commercial
cultivars on MS medium containing TDZ and NAA was assessed. Cultivars Albireo, H3, Polarno white and WSP-3 that were bred from G. scabra as pollen parents showed higher regeneration (80 and 100%), whereas G. triflora cultivars Homo• Ihatovo, Iwate, Iwate-otome and Maciry showed less regeneration (30 to 75%). A comparison of mean numbers of regenerated shoots indicated cv. WSP-3 and Polarno white to have the highest value for this parameter (8-10 shoots per explant). |
| Rooting | Regenerated shoots were transferred to MS medium containing 30 g/L sucrose and 2 g/L gellan gum for rooting. Shoots produced in culture were easily rooted in phytohormone-free medium in four weeks, and then acclimatized. |
| Acclimation | The regenerated plants with a well-established root system were carefully rinsed to remove gellan gum and transferred to pots containing vermiculite. Plants in the pots were then transferred to a greenhouse following completion of acclimatization. |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | Each of twenty regenerated plants grown in a greenhouse were phenotypically normal with respect to leaf shape and growth features during early growth stages. The number of chromosomes was counted in the root tip cells of ten regenerated plants of each cultivar, and was found to be 2n=26, which corresponds to the diploid number for the species. |
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| Extraction | |
| Analitical methods | |
| Notes | G. triflora, interspecific hybrid of G. triflora × G. scabra |