CHAPTER 4: CAULOGENESIS AND RHIZOGENESIS
4.1 MATERIALS AND METHODS
Indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), α-naphthalene acetic acid (NAA), 6-benzyladenine (BA), myo-inositol, vitamins (thiamine HCl, nicotinic acid, pyridoxine HCl and glycine) were obtained from Sigma-Aldrich (St. Louis, USA), agar bacteriological from Oxoid Ltd (Bastingtoke, England), and polyvinlypyrrolidone from BDH Chemicals (Poole, England). Other plant growth regulators, meta-topolin [6-(3-
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hydroxybenzylamino)purine (mT)], meta-topolin riboside [6-(-3-hydroxybenzylamino)- 9-β-D-ribofuranosylpurine (mTR)], and meta-methoxytopolin riboside [6-(-3- methoxybenzylamino)-9-β-D-ribofuranosylpurine (memTR)] were obtained from Olchemim Ltd, Czech Republic. All other chemicals used to prepare growth media were of analytical grade.
4.1.2PLANT MATERIAL
Hypocotyls and epicotyls were obtained from 30-60 day-old 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 90-100 µmol m-2 s-1 provided by cool white fluorescent light (Osram L 58W/640, Germany).
Initial shoot explants were obtained from seedlings raised in plant growth chambers, whilst subsequent shoots were from in vitro cultured explants. Explants from seedlings raised in plant growth chambers were rinsed thoroughly in running tap water, and surface-decontaminated in 70% alcohol for 1 min followed by 2% sodium hypochlorite with Tween 20 (polyoxyethylene sorbitan monolaurate, Saarchem, Krugersdorp, South Africa) for 20 min. The explants were thoroughly rinsed in sterile distilled water 3 times and cultured on MS growth medium supplemented with vitamins and plant growth regulators.
4.1.3MEDIA AND GROWTH CONDITIONS
The basal medium consisting of MS supplemented with vitamins, sucrose (30 g l-1), myo-inositol (0.1 g l-1), polyvinylpyrrolidone (3 g l-1) and plant growth regulators, was used for all the experiments. The medium was adjusted to pH 5.8 with KOH or HCl before adding the gelling agent (8 g l-1 Agar Bacteriological, Agar No.1, Oxoid Ltd, Bastingstoke, England) and autoclaving at 121 °C, 15 KPa for 20 min. The culture tubes contained 10 ml of MS medium for all experiments. Cultures were maintained at a temperature of 25 ± 2 °C under either a 16-h or 24-h photoperiod regime. Unless otherwise stated, 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).
76 4.1.4INDUCTION OF SHOOT ORGANOGENESIS
Four aromatic cytokinins, BA, mT, mTR, and memTR at concentrations of 2.0, 4.0 and 8.0 µM were evaluated for the induction of adventitious shoot formation using shoot explants. A set of cultures without any cytokinin served as the control. Each treatment was replicated twice and each replicate consisted of 18 explants.
Combined data from the two separate experiments were used for statistical analysis.
Data on shoot induction (%), number of adventitious shoots per responding explants, and shoot length were recorded after 4 and 8 weeks in culture under a 16-h photoperiod.
4.1.5SHOOT INDUCTION FROM HYPOCOTYLS AND EPICOTYLS
Hypocotyl or epicotyl segments (10 mm length) were placed horizontally on MS medium supplemented independently with BA, mT, mTR, and memTR at different concentrations (1.0, 4.0 and 8.0 µM). The control consisted of hypocotyl or epicotyl segments without any cytokinin supplementation. The treatments consisted of 15 replicates each and the experiment was repeated twice. The data from the two experiments were combined for statistical analysis. The cultures were maintained under a 24-h photoperiod and data on shoot induction (%), number of adventitious shoots per explant, shoot length and shoot fresh weight were collected after 6 weeks.
4.1.6INDUCTION OF ROOT FORMATION
Adventitious shoots (> 5 mm) were excised from the original explants and transferred to MS medium (as described previously) supplemented with IAA, IBA or NAA at different concentrations (1.0, 2.0, 4.0, 6.0 and 8.0 µM). The control consisted of cultures without auxins. To evaluate the effect of temperature on rhizogenesis, adventitious shoots were cultured on MS medium supplemented with 4.0 µM IBA and maintained at different temperatures (20, 25, 30 and 35 °C) under a 16-h photoperiod. Each treatment was replicated twice and each replicate consisted of 15 explants. The combined data were used for statistical analysis. Data on root induction (%), number of adventitious roots per explant, and root length were recorded after 4 and 8 weeks in culture. The influence of photoperiod on root induction was evaluated on adventitious shoots cultured under 16-h and 24-h
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photoperiods, respectively. The shoots were cultured on MS medium supplemented with 4.0 µM IBA, and data collection was done after 8 weeks. The photoperiod experiment was replicated twice and each treatment consisted of 20 explants.
After 8 weeks in culture, rooted plantlets were planted in plastic containers in a 1:1 (v/v) vermiculite and sand mixture and placed in a controlled environment misthouse for four days for acclimation ex vitro. A high pressure fog system was used to maintain high relative humidity in a range between 90 and 100%. From the misthouse the plantlets were transferred to a greenhouse in which the temperature was maintained at 25 ± 2 °C.
4.1.7STOMATAL ANATOMICAL AND MORPHOMETRIC EVALUATION
Leaves were harvested from all treatments during the light period and immediately immersed in cold 3% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.2.
One disc was cut from each of the four leaves per treatment in a glutaraldehyde solution. Fixation of specimens was done in cold 3% glutaraldehyde in 0.05 M sodium cacodylate buffer and incubated for 24 h at 4 °C as described in section 3.1.5. After the 24 h incubation, the specimens were washed twice for 30 min each in 0.05 M cacodylate, and dehydrated through a graded ethanol series as described in section 3.1.4. The leaf discs were dried in CO2 in an HCP-2 critical point drier (Hitachi, Japan), mounted on aluminium SEM stubs with the abaxial side up and coated with a 20 nm layer of gold-palladium in an Eiko IB-3 ion coater (Ibaraki, Japan). The specimens were viewed in a ZEISS EVO® MA 15 SEM (Carl Zeiss SMT, Germany) fitted with a digital imaging system driven by SmartSEM software and operating at 10.0 kV and a working distance (WD) of between 10.0-10.5 mm.
For morphometric estimation of stomatal density and size, the methods described by MIGUENS et al. (1993) and SNIDER et al. (2009) with modifications were used.
Four disc areas per leaf were imaged at a magnification of 600X, and the number of mature stomata on each image was counted. Each of these values was treated as n = 1, giving a total of four replicates per treatment for statistical analysis. To compare density, diameter and length of stomata, equi-magnified leaf disc areas (454 µm X 306 µm) of 1.38924 X 105 µm2 each were used, and the number of
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stomata was expressed per mm2. The abaxial surface was used for all the measurements as it has been shown to contain more stomata in other plant species (MIGUENS et al. 1993; ZACCHINI et al. 1997; MAJADA et al. 2001).
4.1.8EXPERIMENTATION AND DATA ANALYSIS
Data were recorded after 4 and 8 weeks (unless stated otherwise) on the shoot and root multiplication. The experiments were arranged in a completely randomised design and each experiment was repeated twice. Percentage data were arcsin transformed to validate the one-way analysis of variance (ANOVA) assumption of normality of data distribution (TERESO et al. 2008) and then subjected to statistical analysis. For all the tested parameters ANOVA was used to find differences among treatments, mean values were separated by Duncan Multiple Range Test and significant effects were accepted for p ≤ 0.05. Data computations were done using SPSS for Windows (version 15, SPSS®, Chicago).