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Plants of Drosera adelae were obtained from a local nursery in Penang mainland and propagated vegetatively by root cuttings. The root cuttingss were planted in pure sphagnum moss medium in plastic pots. The young leaves of D. adelae were used as explant and cultured in an enclosure or closed container provided with artificial environment called terrarium. Plants were subjected to 16 hours of artificial lighting using cool white fluorescent light with average light intensity of 35 µmol/ m2/ s and the water level of the terrarium was continuously maintained at 100 mL. The plant material was grown inside the terrarium for four weeks before the leaf explants were used for the establishment of aseptic explants.
3.2 Establishment of Aseptic Leaf Explants and Induction of Shoot Formation
Two different protocols were used to establish the aseptic leaf explants. In the first protocol, the young leaves of D. adelae excised from mother plant were washed thoroughly using running tap water. The leaves were brushed for 10 minutes interval using painting brush with 10% commercial detergent solution. The leaves were then immersed under 70% ethanol for 10 seconds and surface sterilized using double stage sterilization method. First stage of sterilization was carried out using 5% Clorox®, a commercial bleach solution (5.3% sodium hypochloride, NaOCl) with 2-3 drops of Tween 20 for five minutes followed by rinsing three times with sterile distilled water for three minutes each time. The leaves were again surface sterilized with 3% Clorox® for five minutes for second stage. After rinsing three times with sterile distilled water, the leaves were cut into the size of 1.0 - 1.0 cm2 pieces. Each leaf explant was inoculated into 100 mL Erlenmeyer flask containing 25 mL sterile distilled water.
In the second protocol, the leaf explants were washed thoroughly using running tap water. They were brushed for 10 minutes using painting brush with 10% commercial detergent solution then subsequently surface-sterilized with 70% ethanol for 10 seconds followed by 5% Clorox® solution with 2-3 drops of Tween 20 for 5 minutes. After rinsing three times with sterile distilled water, the leaf explants were further surface-sterilized using the same procedure. The leaf explants were then inoculated into 100 mL Erlenmeyer flask containing 25 mL sterile distilled water. The sterile distilled water in 100 mL Erlenmeyer flask was autoclaved at 121°C at 1.05 kg cm-2 for 11 minutes using Tomy SS-325 Autoclave.
Total of twelve replicates were used for each protocol. Percentage of aseptic leaf explants and percentage of survival were determined after three weeks of culture. The number of shoots produced from each leaf explants were recorded after five weeks of culture. The data were analyzed using Student t-test at p ≤ 0.05. The data for the percentage of aseptic leaf explants establishment and the percentage of survival were recorded.
3.3 Effect of Agitation on Shoot Induction.
The aseptic leaf explants established with double stage surface sterilization protocol 1 were cut into the size of 1.0 - 1.0 cm2 pieces. The leaf explants were inoculated into 100 mL Erlenmeyer flask containing 25 mL sterile distilled water. 12 replicates with one leaf explants per replicate were kept in stagnant condition while another twelve were agitated on Labwit ZHWY-3112 orbital shaker at 115 rpm. The number of shoots produced per explants was determined after five weeks of culture. The data were analyzed using Student t-test at p ≤ 0.05.
3.4 Effects of MS Strength on Growth of Plantlets
Five weeks old in vitro shoots derived from the leaf explants were removed from the leaf explants. They were inoculated into 250 mL glass culture bottle containing 25 mL different strength of Murashige and Skoog (MS) medium (1962) (full strength MS, ½ MS, and ¼ MS) supplemented with 30 g/L sucrose, 8.0 g/L of agar (Algas, Chile). The medium was adjusted to pH 5.7 - 5.8 prior to autoclave.
Seven replicates were used for each MS strength and the experiment was repeated twice. The number of leaves and roots, leaf length, and root length were determined after eight weeks of culture. Data obtained were analyzed using one-way analysis of variance (ANOVA) and the best MS strength was determined using Duncan's multiple range test at p = 0.05.
Eight weeks-old rooted in vitro plantlets of D. adelae were removed from the culture medium and washed thoroughly under running tap water to remove the remaining gelling agent, agar. The plantlets were transferred into square plastic trays (20 cm - 20 cm) containing pure long sphagnum moss overlaid with tap water placed under greenhouse conditions. The container was covered with plastic sheet for one week. Plastic cover with holes was used to cover the trays for second and third weeks. The temperature was maintained constantly for 4 weeks (28 ± 2 °C during day time and 24 ± 2 °C during night time). The percentage of surviving plantlets was recorded after four weeks of acclimatization period.
3.6 Morphological Studies of Drosera adelae Leaves
3.6.1 Light Microscopy
The in vivo and in vitro leaves of D. adelae were cut into small pieces and fixed in formalin: acetic acid: ethanol (FAA) solution (1:1:18). Samples that had been fixed were rinsed with distilled water five times. The samples were then dehydrated by passing through a tertiary butyl alcohol- xylol series and embedded in paraffin wax. The specimens were cut using rotary microtome into blocks and serial sections (10-12 µm). The microtome sections were mounted onto slides and allowed to dry for 24 hours before staining with safranin and fast green. Cover slips were mounted and dried with histoclad mounting medium. The permanent slides were observed under light microscope fitted with a camera (Olympus Bx50) at 4 - to 40 - magnification.
3.6.2 Scanning Electron Microscopy
Leaves of D. adelae were cut into small pieces (0.5cm x 0.5cm), the leaf samples were then fixed overnight in McDowell-Trump fixative prepared using 0.1 M phosphate buffer or cacoadylate buffer (pH 7.2) at 4°C. The leaves were then washed with 0.1 M phosphate buffer for 30 min and post fixed in 1% Osmium tetroxide (OsO4) solution at 25°C for one to two hours. The specimens were then rinsed with distilled water for 20 minutes and continued with dehydration process using 50% ethanol, 75% ethanol, 95% ethanol and 100% ethanol. The dehydrated tissues were then immersed in hexathyldisilazane (HMDS) for 10 min. HMDS from the specimen vial was decanted and the specimen vial with the tissue was left in the desiccators to air dry at room temperature. The specimens were then mounted by double adhesive tape on aluminium stubs, and coated with thin gold layer (40 to 60 mm) using Bio-Rad SEM coating system. Leaf morphology was examined with the aid of Scanning Electron Microscope (SEM) at 8 to 10 kV. The morphology of stomata, glandular, and non-glandular trichomes were observed and documented using Soft Image System (SIS) programme camera.