MATERIALS AND METHODS

CHAPTER 3: IN VITRO MORPHOGENESIS OF LEAF-DERIVED NODULAR

3.1 MATERIALS AND METHODS

Leaf explants were obtained from S. birrea seedlings grown in plant growth chambers (Controlled Environments Ltd, Manitoba, Canada) at 25 ± 2 °C under a 16-h photoperiod at a photosynthetic photon flux density of 100 µmol m^-2 s^-1 provided by cool white fluorescent light. The explants were rinsed thoroughly in running tap water, before being surface-decontaminated in 70% alcohol for 1 min

59

followed by 2% sodium hypochlorite for 20 min with a few drops of Tween 20 (polyoxyethylene sorbitan monolaurate, Saarchem, Krugersdorp, South Africa).

The explants were then thoroughly rinsed in sterile distilled water 3 times. Leaf explants were cultured with the abaxial surface on the growth medium.

3.1.2INDUCTION OF NODULAR MERISTEMOIDS

Woody plant medium (WPM; LLOYD and McCOWN 1981) and Murashige and Skoog medium (MS; MURASHIGE and SKOOG 1962) were supplemented with vitamins, sucrose (30 g l^-1), myo-inositol (0.1 g l^-1), polyvinylpyrrolidone (PVP) (3 g l^-1) and plant growth regulators and adjusted to pH 5.8 before the addition of the gelling agent (8 g l^-1 Agar Bacteriological, Agar No.1, Oxoid Ltd, Bastingstoke, England). The media were then autoclaved at 121 °C, 15 KPa for 20 min. The cytokinin, 6-benzyladenine (BA), was used in different concentrations (0, 1.0, 2.0 and 4.0 µM) in combination with three auxins, indole-3-acetic acid (IAA), indole-3- butyric acid (IBA) and α-naphthalene acetic acid (NAA) at final concentrations in the medium of 0, 1.0, 2.0 and 4.0 µM. Plant growth regulators were added to the growth medium before autoclaving. All the plant growth regulators were obtained from Sigma-Aldrich, St. Louis, USA. Cultures were maintained at a temperature of 25 ± 2 °C under a 16-h photoperiod. A constant photosynthetic photon flux density of 40 µmol m^-2 s^-1 was provided by cool white fluorescent light (Osram L 58W/640, Germany). Light intensity was measured with a quantum radiation sensor (Model Skp 215, Skye Instruments Ltd, Llandridod Wells, Powys, UK). Each treatment consisted of 18 replicates and the experiment was done twice. After 4 weeks in culture data on the percentages of explants that formed nodules, callus and differentiated shoot buds, as well as the mean number of shoots per responding explant were recorded.

3.1.3PLANTLET REGENERATION

For plantlet development nodular meristemoids were maintained on solid medium or transferred to liquid plantlet development medium consisting of MS medium supplemented with vitamins, sucrose (30 g l^-1), myo-inositol (0.1 g l^-1), polyvinylpyrrolidone (3 g l^-1), and combinations of BA and NAA or IBA that gave the best results in the culture initiation experiments. The nodules were incubated

60

at 25 ± 2 °C under a 16-h photoperiod at a photosynthetic photon flux density of 40 µmol m^-2 s^-1. Nodular meristemoids in liquid medium were placed on a shaker at a speed of 120 rpm. Sixteen replicates were done for each treatment in liquid shake culture, and the experiment was repeated twice. The liquid shake culture treatments consisted of eight flasks per treatment with two clusters of nodular meristemoids in each. After 4 weeks plantlet growth was recorded. Data on the number of differentiated shoots and mean shoot length were recorded after a 4- week culture period.

3.1.4HISTOLOGICAL EXAMINATION

Nodular meristemoids were fixed overnight at 4 °C in 3% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2. After rinsing with buffer (twice, 30 min each), the embryos were post-fixed in 2% osmium tetroxide in 0.05 M sodium cacodylate buffer for 10 min (JAYASANKAR et al. 2003). After rinsing twice (30 min each time) in the same buffer solution, the samples were serially dehydrated (10 min each time) in ethanol (30–90%) and three times in 100%. The specimens were infiltrated with propylene oxide (twice for 30 min each), and embedded with a serial increment of Epon/Araldite resin: propylene oxide [25:75, 50: 50, 75:25 and 100: 0 (v/v)]. The samples were polymerised in 100% Epon/Araldite resin and polymerised at 70 °C for 48 h. Blocks of resin, approximately 1–4 mm³, containing an embryo or cluster of embryos were excised and attached to the solidified resin blanks with superglue (JAYASANKAR et al. 2003). Sections were then cut on an LKB ultrotome III^® (Stockholm, Sweden) using glass knives. Sections (5 µm thick) for light microscopy were stained with Ladd Multiple stain (Toluidine blue and basic fuchsin in 30% ethyl alcohol) for 2 min. Excess stain was washed away with distilled water and sections were secured to glass slides with gentle heat at 70 °C.

Sections were immediately observed under a fluorescence microscope (Olympus AX70, Olympus, Japan) equipped with a digital capture system with a minimum 300 dpi resolution.

3.1.5MACROSCOPIC EVALUATION

Macroscopic images were recorded using a stereomicroscope (Leica MZ16, Switzerland) fitted with a digital image capture system with a minimum resolution

61

of 300 dpi (GOMES et al., 2006). Samples for scanning electron microscopy were fixed in cold 3% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2 and incubated overnight at 4 °C. The nodules were washed twice at 30 min each time in the same buffer, and dehydrated through a graded ethanol series as described earlier. The samples were critical point dried with carbon dioxide using a HCP-2 critical point dryer (Hitachi, Japan), mounted on aluminium stubs and sputter- coated with a 20 nm layer of gold-palladium. The specimens were immediately examined with a Hitachi S–570 scanning electron microscope (Hitachi, Japan) operating at 7–8 kV.

3.1.6EXPERIMENTATION AND DATA ANALYSIS

Data were recorded after 4 weeks on the frequency of nodular meristemoid induction, shoot regeneration and callus formation. In addition, some of the nodular meristemoids were maintained on the same medium or re-cultured in liquid medium and evaluated for shoot regeneration after 4 weeks. The experiments were arranged in a completely randomised design and each experiment was repeated twice. Percentage data were arcsin transformed before being statistically analysed. One-way analysis of variance (ANOVA) was done and Tukey‟s test was used to separate differences among treatment means (p ≤ 0.05). Data were analysed using SPSS for Windows (version 15, SPSS^®, Chicago).