| Plant latin name | Gentiana scabra Bunge，Gentiana manshurica Kitagawa |
| Literature code | Gentiana_scabra-Ref-1 |
| Reference | Huang S-H et al., Botanical Studies: 55:56 ( 2014) |
| Summary | In the present study, we report an efficient micropropagation system. Shoot apices of six weeks old in vitro grown G. scabra plants were used as explants for the in vitro propagation. Induction of multiple shoots (9.1/explant) was achieved on the culture of shoot apices on half strength Murashige and Skoog’s basal medium (MSBM) containing 2.0 mg/L 6-benzylaminopurine (BA), 3% sucrose and 0.9% Difco agar. In vitro shoots induced profuse rooting on half strength of MSBM supplemented with 0.1 mg/L 1-naphthaleneacetic acid (NAA), 3% sucrose and 0.3% gelrite. A two-stage ventilation closure procedure during the in vitro culture, and transparent sachet technique enhanced the survival rate of G. scabra plantlets to 96% in the greenhouse. Tissue culture plants flowered after 5 months of transfer to pots. |
| Objectives | The main objective of the study was to develop a simple and an efficient in vitro propagation method of G. scabra with a high survival rate of plantlets. |
| Materials | In vitro plants of G. scabra obtained from the Taiwan Sugar Corporation, Taiwan |
| Explant | |
| Initial culture | |
| Shoot multiplication | In our initial experiments on different strengths (1, 1/2 and 1/4) of MS medium, it was observed that
1/2 MS medium resulted in the better shoot/root growth in comparison to 1 or 1/4 strengths, hence, 1/2 MS medium was used for all the experiments. Between the two cytoknins, BA was more effective and induced a higher number of shoots per explant compared to Kin. The maximum average number of multiple shoots (9.1 shoots/explant) could be induced in shoot apices on 1/2 MS supplemented with 2.0 mg/L BA, 3% sucrose and 0.9% agar, followed by 8.8 shoots/explant on medium with 1.0 mg/L BA. There was an inverse correlation between concentrations of both BA and Kin and lengths of induced shoots. Average shoot elongation was higher (2.82 and 3.56 cm) at lower concentrations of BA (0.1 mg /L) and Kin (0.1 mg /L), respectively. |
| Rooting | On culture of in vitro derived shoots on 1/2 MS medium supplemented with a range of concentrations of two auxins (NAA and 3-indolebutyric acid: IBA) for 8 weeks, it was observed that the lower concentrations of NAA or IBA resulted in a higher number as well as better root/shoot growth. NAA at 0.1 mg/L induced the greater number of roots (37.2) and fresh weight (1.55 g) in comparison to IBA at 0.1 mg/L which induced a lesser number of roots (25.8) and lesser fresh weight (1.20 g). Higher concentrations of IBA and NAA (1.0 mg/L) reduced the number and length of roots as compared to 0.1 mg/L concentration. In contrast to culture obtained from Taiwan Sugar Corporation, roots induced in the MS medium supplemented with NAA 0.1 mg/L were multifold and robust. A supplement of 1.0 mg/L NAA in the MS stimulated callus induction, a trait undesirable for health of roots/shoots and ex vitro survival of plantlets.
Between the two sets of ventilation closure treatments, i.e. one-stage and two-stage with a total incubation period of 8 weeks, overall, all the root/shoot growth parameters including the plant survival percentage were higher with two-stage ventilation closure treatments. There were significant differences in root/shoot growth parameters among the treatments within the one-stage, and twostage ventilation closures itself.
Among the one-stage ventilation closure treatments,the maximum number of roots (20.20), root length (4.58 cm), shoot length (2.23 cm), fresh weight (1.09 g) and plant survival percentage (94.44) was achieved with 2AF8wk, i.e. when culture vessels were closed with 2 layers of aluminum foil and incubation for 8 weeks. In contrast to aluminum foil, there was an overall decrease in root/shoot growth parameters when culture vessels were closed with 2, 3 and 4 dispense papers. Among the three DP treatments, the maximum number of roots (18), root length (3.78 cm), shoot length (1.51 cm), fresh weight (0.94 g) and plant survival percentage (91.67) was achieved with 4DP8wk, i.e. when culture vessels were closed with 4 layers of dispense paper and incubation for 8 weeks. There was no drastic difference in root/shoot growth parameters and plant survival percentage in the ventilation closure treatment 4H2AF8wk, i.e. 2AF with 4 holes punched in the center and covered with air permeable tape. Among the 4 two-stage ventilation closure treatments, the maximum number of roots (33.7), root length (5.89 cm), fresh weight (1.41 g) and plant survival (96%) were obtained with treatment 2AF4wk/4DP4wk, i.e. when culture vessels were closed with 2 AF for 4 weeks and then AF replaced by 4DP for the next 4 weeks. |
| Acclimation | After 8 weeks of incubation under the ventilation closure conditions, the plantlets were carefully taken out of culture vessels and rinsed gently with running tap water to remove traces of agar. The plantlets were then blotted dry on paper towel and their fresh weights, number of shoots and roots, length of roots and shoots were recorded. Thereafter, plantlets were briefly treated with 0.1% Benlate (a systemic fungicide) solution (DuPont, Wilmington, DE) and transplanted into plastic pots containing a mixture of peatmoss:perlite:vermiculite (2:1:1 v/v). For acclimatization, each potted plant was covered with a transparent polyethylene sachet. After one week, a small hole was made in a corner of each sachet, and another hole was made in the opposite corner after 2 weeks. The top corners on both sides of each sachet were cut open after 3 weeks, and sachets were completely removed after 4 weeks. Thereafter, these were shifted to the University’s greenhouse. The plants were irrigated every day with tap water. The plant survival against each ventilation closure treatment was recorded after 2 months.
Between the one-stage and two-stage ventilation closure treatments, the overall survival percentages were higher (90–96) in the two-stage treatments. The maximum survival percentage (96) was recorded with the twostage treatment 2AF4wk/4DP4wk, i.e. culture vessel closed with 2 layers of aluminum foil for first 4 weeks and then AF replaced with 4 layers of disense paper. Covering of plants with transparent polyethylene sachets for the first 4 weeks and its gradual exposure to the ambient conditions was supportive of acclimatization process and gave rise to higher plantlet survival percentages. Tissue culture plants flowered in the greenhouse after 5 months of their transfer to pots. |
| Planting | |
| Cultivation conditions | |
| Traints of regenerants | |
| Ingredients analyzed | |
| Extraction | |
| Analitical methods | |
| Notes | |