| Plant latin name | Chrysanthemum morifolium Ramatulle |
| Literature code | Chrysanthemum_morifolium-Ref-2 |
| Reference | Shatnawi M. et al, Jordan Journal of Biological Sciences, 3(3): 101-110 (2010) |
| Summary | A micropropgation method by multiple shoot formation of Chrysanthemum morifolium has been developed. Explants growing in greenhouse were used to establish cultures of C. morifolium. Shoot tips were surface sterilized and cultured on Murashige and Skoog (MS) media. Successful in vitro multiplication of chrysanthemum was achieved on MS medium supplemented with benzyl amino purine (BAP) at 0.3 mgl-1 . In vitro rooting was successfully achieved on MS media supplemented with different concentration of auxins. The in vitro response to salinity stress (0 to 300 mM NaCl) was also tested. Shoot proliferation was gradually reduced at higher NaCl concentrations. Shoot length, number of leaves, fresh and dry weight, chlorophyll, and carotenoid decreased with elevated salinity concentration. Proline and sodium contents increased with elevated salinity, whereas potassium, nitrogen and protein content decreased. Plantlets grown in vitro presented tolerance and their growth was negatively affected at high salt concentrations. Elevated salinity significantly reduced microshoot protein. It is concluded that in C. morifolium response to in vitro salinity stress may provide a system for production under field conditions. |
| Objectives | The aim of this study was, to initially establish an effective way for in vitro proliferation method for C. morifolium Ramat and furthermore to investigate the in vitro response of C. morifolium when being subject to NaCl stress. |
| Materials | Chrysanthemum morifolium growing in greenhouse |
| Explant | Shoot-tips |
| Initial culture | Microshoots of C. morifolium Ramat, (Balady) shoot tips were washed under running tap water and then sterilized with 70% ethanol for 30 seconds, then dipped in 3.5% sodium hypochlorite for 15 minutes. Finally, excess detergent was removed by rinsing in sterile distilled water four times each for five minute, under the laminar air-flow cabinet.
Medium was solidified using 8.0 gl-1 agar agar, then dispensed in test tubes (8 ml each), and autoclaved at 121
°C. Shoot tips were grown on solid half strength MS medium. Cultures were maintained in the growth chamber at 24 ± 2 °C and 16 h light/8 h dark. Microshoots were then subcultured on full strength MS medium for six times to have enough mother stock prior to experiments initiation. The pH of MS medium was adjusted to 5.8. Medium was solidifying 8.0 gl-1 agar agar, dispensed in flasks (60 ml), and autoclaved at 121°C. Cultures were maintained in the growth chamber at23±2°C and16h lights(50μmolm-2s-1)/8dark. |
| Shoot multiplication | Microshoots were subcultured to hormone-free MS medium for two weeks to eliminate any carry-over effects of the basic cytokinin. For shoot proliferation, microshoots (15 mm in length) were subcultured to MS medium supplemented with either, benzyl amino purine (BAP) or kinetin, at 0.0, 0.3, 0.6, 0.9, 1.2 and 1.5 mgl-1. The pH of MS medium was adjusted to 5.8. Medium was solidified using containing 8 gl-1 agar and 30 gl-1 sucrose supplemented to the medium. 60 ml/flask of the MS medium was dispensed into 250 ml Erlenmeyer flasks and autoclaved at 121 °C. Explants were incubated in a growth room under 16 h light/8 h dark (50 μmol m-2s-1). After six weeks growth periods, data were collected on shoot length and number of shoot per explants. Increasing BAP from 0.0 to 0.3 mgl-1 increased the number of proliferated shoots from 1.98 to 4.35 (P=0.05). Number of proliferated shoots at 1.5 mgl-1 BAP was lower compared to those at 0.3 mgl-1 BAP. Maximum shoot production was obtained on a medium containing 0.3 mgl-1 BAP. Similarly, kinetin increased average number of shoots/explants. Increasing concentration of BAP or kinetin decreased shoot length. |
| Rooting | Microshoots were subcultured to hormone-free MS medium for 2 weeks to eliminate any carry-over effects of the cytokinin. Microshoots (15 mm in length) were subcultured on MS medium supplemented with (0.0, 0.2, 0.4, 0.6, 0.8, or 1.0 mgl-1) IBA (indole-3-butyric-acid), IAA (indole-3-acetic-acid) or NAA (1- naphthalene acetic acid containing 8 gl-1 agar and 30 gl-1 sucrose. Explants were incubated in a growth room under 16 h light/8 h dark (50 μmol m-2s-1). Data were collected after six weeks growth period on number of roots, roots length, and number of leaves. Microshoots were successfully rooted in vitro on MS medium supplemented with 0.0, 0.2, 0.4, 0.6, 0.8 or 1.0 mgl-1 of IBA, IAA or NAA. Root formation start after 14 days growing period. Rooting occurred in bases of shoots growth on solid media supplemented with IBA, IAA, or NAA. No callus formation appeared at the bases of the cuttings. Increasing IBA, IAA or NAA concentrations resulted in a significant effect on root length. Maximum root number was obtained with the addition of IBA at 0.2 mgl-1, with an average of 18.75 roots per microshoot. Root length was significantly decreased with the use of IBA, IAA and NAA at 1.0 mgl-1. Increasing IBA concentrations significantly increased number new leaves formation. Maximum number (17.07) of leaves was obtained at 0.4 mgl-1 IAA, followed by 14.75 at 0.4 mgl-1 IBA. |
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