CHAPTER 3: RESULTS
3.5 Viabilty of embryonic axes of T. dregeana in storage after alginate gel
3.5.1 Alginate gel encapsulation, storage and assessment under hydrated conditions
Nipastat (the mixture of methyl-, butyl-, ethyl-, propyl- and isobutyl-paraben) and NaDCC (sodium dichloro-isocyanurate), both of which were highly effective in inhibiting proliferation of the test isolates, were used to decontaminate embryonic axes of T. dregeana before encapsulation and storage at 16°C (Fig. 3.22).
Figure 3.22: Embryonic axes of T. dregeana encapsulated with alginate gel and stored hydrated on a grid in a magenta box.
Prior to alginate coating, embryonic axes were immersed in Nipastat at 0.2% for 10 min or NaDCC at 0.2% (2000 ppm) for 20 min and then stored on grids in magenta boxes. However, after 14 d storage, fungal contamination was seen to have persisted, the major contaminant being Penicillium spp. for axes treated with both Nipastat (Tables 3.37 and 3.38) and NaDCC (Tables 3.39 and 3.40) prior to alginate encapsulation. Additionally, the embryonic axes had not retained viability (Fig. 3.23), whether treated with Nipastat, NaDCC, or distilled water (control). It was considered that axis viability loss could have been due to the absence of a carbon source. When Nipastat was used as the decontaminant (Tables 3.37 and 3.38), persistence of the contaminants was greatly reduced in the encapsulated axes compared with those exposed to distilled water prior to encapsulation.
Unfortunately, the concentration of Nipastat could not be increased above 0.2% because of apparent toxicity/inhibitory effects on the embryonic axes. However, survival of the embryonic axes (Fig.
3.24A) was increased by incorporating 0.5% sucrose in the alginate, as was the case when NaDCC was used for decontamination (Table 3.39). In the case of NaDCC, persistence of the contaminants was resolved by increasing the concentration to 0.03% (3000 ppm) for the same time (20 min) and viability retention (as indicated by germinability after 14 d storage) was enhanced, all axes remaining germinable (Fig. 3.24B) when 0.5% sucrose was incorporated in the alginate gel (Tables 3.39 and 3.40). Although the viability of axes so-encapsulated was lost after 28 d in storage, this clearly was not associated with residual fungal contamination (Table 3.39).
Table 3.37: Viability and contamination after storage of axes of T. dregeana encapsulated in alginate gel (incorporating sucrose or not) after treatment for 10 min with 0.2% Nipastat, (n = 30). Mean values followed by the different lower case letters within columns and different uppercase letters in each row of individual parameters (Germination/Contamination) between different time intervals (14, 28 & 42 d) are significantly different. (Dependent variables were subjected to Arcsine transformation, two-way ANOVA, Duncan’s Multiple Range Test, p ≤ 0.05).
Treatment with:
Sucrose (%)
% Germination of axes assessed after 14, 28 and 42 d in culture
% Axes showing contamination
14 28 42 14 28 42
Distilled H2O (control)
0.0 0.0aA 0.0aA 0.0aA 40.0aA 60.0aB 60.0aB
0.5 60.0bA 0.0aB 0.0aB 46.7aA 66.7aB 66.7aB
Nipastat
0.0 0.0aA 0.0aA 0.0aA 13.3bA 26.7bB 33.3bC 0.5 86.7cA 0.0aB 0.0aB 13.3bA 20.0bB 26.7bC
Table 3.38: Contaminants isolated after storage for 14, 28 & 42 d of embryonic axes of T.
dregeana encapsulated in alginate (incorporating sucrose or not) after treatment with 0.2%
Nipastat for 10 min, (n = 30). Mean values followed by the different lower case letters within columns for each isolate between different treatments and different uppercase letters in each row are significantly different. (Dependent variables were subjected to Arcsine transformation, two-way ANOVA, Duncan’s Multiple Range Test, p ≤ 0.05).
Treatment with:
Sucrose (%)
Contaminant % Axes showing contamination
14 d 28 d 42 d
Distilled H2O (control)
0
Pencillium spp.
40.0aA 56.7aB 46.7aC
0.5
40.0aA 53.3aB 66.7bB
Nipastat 0 26.7bA 26.7bA 33.3cA
0.5
13.3cA 20.0bB 26.7cC
Distilled H2O (control)
0
Fusarium spp.
0.0aA 13.3aB 13.3aA
0.5
0.0aA 6.7aB 0.0bB
Nipastat 0 0.0aA 0.0bA 0.0bA
0.5
0.0aA 0.0bA 0.0bA
Distilled H2O (control)
0
Bacteria
0.0aA 0.0aA 0.0bB
0.5
6.7bA 6.7bA 0.0aA
Nipastat 0 0.0aA 6.7bB 0.0aA
0.5 0.0aA 0.0aA 0.0aA
Table 3.39: Viability and contamination after 14, 28 & 42 d of axes of T. dregeana encapsulated in alginate (incorporating sucrose or not) after treatment with 0.2% or 0.3%
NaDCC for 20 min. Control axes were exposed to distilled water before encapsulation, (n = 30). Mean values followed by the different lower case letters within columns and different uppercase letters in each row of individual parameters (Germination/Contamination) between different time intervals (14, 28 & 42 d) are significantly different. (Dependent variables were subjected to Arcsine transformation, two-way ANOVA, Duncan’s Multiple Range Test, p ≤ 0.05).
Treatment with:
Conc: Sucrose (%)
% Germination of axes % Axes showing contamination
14 d 28 d 42 d 14 d 28 d 42 d
Distilled H2O (control)
0.0 0.0aA 0.0aA 0.0aA 33.3aA 60.0aB 60.0aB 0.5 53.3bA 0.0aB 0.0aB 46.7bA 60.0aB 66.7aC
NADCC
0.2 0.0 0.0aA 0.0aA 0.0aA 20.0cA 33.4bB 46.6bC 0.5 73.3cA 0.0aB 0.0aB 26.7cA 47.0cB 47.0bB 0.3 0.0 0.0aA 0.0aA 0.0aA 0.0dA 0.0dA 10.0cB
0.5 100.0dA 0.0aB 0.0aB 0.0dA 0.0dA 13.3cB
Table 3.40: Contaminants isolated after storage for 14, 28 & 42 d of embryonic axes of T.
dregeana encapsulated in alginate (incorporating sucrose or not) after 20 min treatment with 0.2 or 0.3% NaDCC, (n = 30). Mean values followed by the different lower case letters within columns for each isolate between different treatments and different uppercase letters in each row are significantly different. (Dependent variables were subjected to Arcsine transformation, two-way ANOVA, Duncan’s Multiple Range Test, p ≤ 0.05)
Treatment with:
Sucrose (%) Contaminant % Axes showing contamination
14 d 28 d 42 d
Distilled H2O (control)
0.0
Pencillium spp.
40.0aA 53.3aB 66.7aC
0.5 33.3aA 53.3aB 60.0aC
NADCC (0.2%)
0.0 26.7bA 26.7bA 33.3bB
0.5 20.0bA 33.3bB 40.0bC
NADCC (0.3%)
0.0 0.0cA 0.0cA 10.0cB
0.5 0.0cA 0.0cA 13.3cB
Distilled H2O (control)
0.0
Fusarium spp.
0.0aA 6.7aB 0.0aA
0.5 0.0aA 0.0bA 6.7bB
NADCC (0.2%)
0.0 0.0aA 0.0bA 0.0aA
0.5 0.0aA 0.0bA 0.0aA
NADCC (0.3%)
0.0 0.0aA 0.0bA 0.0aA
0.5 0.0aA 0.0bA 0.0aA
Distilled H2O (control)
0.0
A. niger
0.0aA 0.0aA 13.3aB
0.5 0.0aA 6.7bB 0.0bA
NADCC (0.2%)
0.0 0.0aA 0.0aA 13.3aB
0.5 6.7bA 13.3cB 6.7cA
NADCC (0.3%)
0.0 0.0aA 0.0aA 0.0bA
0.5 0.0aA 0.0aA 0.0bA
Distilled H2O (control)
0.0
Bacteria
6.7aA 6.7aA 0.0aB
0.5 0.0bA 0.0bA 0.0aA
NADCC (0.2%)
0.0 0.0bA 6.7aB 0.0aA
0.5 0.0bA 0.0bA 0.0aA
NADCC (0.3%)
0.0 0.0bA 0.0bA 0.0aA
0.5 0.0bA 0.0bA 0.0aA
Figure 3.23: (A) Necrotic appearance after 14 d of embryonic axes coated with alginate gel, but not provided with sucrose; (B & C) embryonic axes, none of which germinated, showing contamination when plated on half strength MS medium in 90 mm Petri dishes. Axes had been decontaminated with Nipastat (B) and distilled water control (C) prior to encapsulation.
Figure 3.24: Seedlings developed after 3 months on half strength MS medium in 90 mm Petri dishes from embryonic axes treated with Nipastat (A) or NaDCC (B) which was stored encapsulated in alginate gel incorporating 0.5% sucrose for 14 d.
3.5.2 The effect of the use of different storage containment on alginate-encapsulated axes of