RESUMO
In this study, an efficient micropropagation protocol was developed for A. longiloba and the antioxidant properties of field-grown plant, in vitro-derived greenhouse-grown plant and in vitro-derived callus extracts were compared. The A. longiloba seeds tested using tetrazolium chloride salt exhibited 89% viability. Due to poor germination capacity of A. longiloba seeds, the seeds were treated with gibberellic acid (GA3) or sulfuric acid (H2SO4). The maximum seed germination of 87% was observed at 30% H2SO4 treatment after 19.00 d, whereas GA3 treatment showed maximum germination of 53% after 22 d. In vitro shoot multiplication was carried out using various types of cytokinins alone or in combination with auxin. Among them, 6-benzyl amino purine (BAP) single treatment was found to be the best hormone. The highest shoot-length (7.26 cm) and maximum number of shoots per explant (18) were recorded at 3-mg L-1 BAP. For in vitro rooting, indole-3-acetic acid at 0.5-mg L-1 was found to be the optimum concentration. Callus was induced using various types of auxins alone or in combinations with cytokinins. The highest percentage of callus of 91 and fresh weight of 6 g was obtained with 3-mg L-1 IAA. The plantlets produced in the current study were subjected to acclimatization. The combination of topsoil and peat moss at 1:2 ratio was found to be the best soil media. In this study, in vitro-derived callus extract showed the highest phenolic content (538 mg GAE), followed by extracts of field-grown plant parts, i.e., fruit and petiole (504 and 300 mg GAE) while in vitro plant extract showed the lowest (98 mg GAE). Meanwhile, the highest flavonoids was recorded in petiole extract. Comparative antioxidant activity study shows, in vitro-derived callus exhibited better DPPH-radical-scavenging activity (IC50: 0.113-mg mL-1) whereas the extracts of petiole, fruit and in vitro plant showed 0.126-, 0.137- and 0.173-mg mL-1, respectively. At the same time, the fruit extract showed better (IC50: 0.088-mg mL-1) ABTS radical scavenging activity than all extracts tested. In conclusion, the in vitro-derived callus extract could be favored for high TPC and better DPPH scavenging activity. Hence, the present study was conducted to establish an efficient micropropagation protocol and to compare the antioxidant activity of the field-grown plant, in vitro plant and in vitro derived callus extracts of A. longiloba.
RESUMO
Dendrobium Sabin Blue is an important orchid hybrid that has been grown extensively as cut flower, potted plant and is also popular for its deep purplish blue flowers. The most efficient long term conservation method of this hybrid is through cryopreservation. Cryopreservation involving the vitrification method consists of explants exposure to highly concentrated cryoprotective solution followed by freezing rapidly in liquid nitrogen. However, these treatments involved highly concentrated cryoprotectant that could incur toxicity to the explants. Hence, cryopreservation protocol requires biochemical analyses in understanding the damages or injuries occurred during cryopreservation treatments. In this study, biochemical analyses revealed a general reduction in chlorophyll, carotenoid and porphyrin content to 0.40 µg/g F W (thawing stage), 31.50 µg/g F W unloading stage and 2230.41 µg/g F W (thawing stage), respectively in comparison to the control treatments. In addition, increased level in proline content were obtained at different cryopreservation stages with highest level (5.42 µmole/g F W) recorded at the PVS2 dehydration stage. Fluctuated outcomes were obtained in catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) enzyme activities in PLBs exposed to different cryopreservation stages. Lowest values recorded for CAT enzyme activity were obtained at the dehydration stage (3.94 U/g). Lowest POX enzyme activities were obtained at the dehydration (122.36 U/g) and growth recovery (106.40 U/g) stages. Additionally, lowest APX enzyme activities values were recorded at the thawing (7.47 U/g) and unloading (7.28 U/g) stages. These have contributed to low regeneration of Dendrobium Sabin Blue protocorm like bodies (PLBs) following cryopreservation. Hence, in the future experimental design, exogenous antioxidant could be included in the cryopreservation procedures to improve the existing protocol.
