Stage 1: At this Stage, decontamination procedure, size of explant, medium composition and culture environment are factors that determine success in
2.2 Materials and Methods
2.2.1 Plant material
The seeds used in this study were obtained from Areka Agricultural Research Centre, Ethiopia. The stored seeds were collected in February and March 1996 from two cultivated genotypes (Mariya and Oniya) ofE. ventricosum, sun dried and stored in brown paper bags at room temperature until used. The studies were
carried out between September 2001 and August 2002. Seeds of two wild types (W01 and W02) of E. ventricosum were collected in January 2003 and the studies on in vitro culture of embryos from these seeds were executed in March 2003. As these seeds were only kept for about 3 months before use they were not considered as having been stored and are referred to as seeds from wild enset types.
2.2.2 Germination of intact stored seeds
Before the in vitro culture of embryos, germination of intact stored seeds was examined. Intact seeds were soaked in hot water (40 °C) for 30 h, as described by TESFAYE (1992) without scarification. Their germination was tested under three different sets of conditions: seeds were planted in pots with sand as medium; then placed in petri dishes on wet filter paper; and thereafter in jars on MS medium supplemented with sucrose (30 g
r
l) and gelled with agar (8 gr
l).2.2.3 Invitro culture of embryos
Different experiments on in vitro zygotic embryo germination were conducted. In all . the experiments, only seeds that sunk when placed in water were used. The seed coat (Figures 2.1 and 2.2) was ruptured using sterile pliers, holding seeds between thumb and forefinger. The embryos, which usually occur in the micropylar area (Figure 2.1), were removed with a scalpel and inoculated onto the medium. The basal medium of MURASHIGE and SKOOG (1962) was used supplemented with sucrose (30 g
r
l) and gelled with agar-agar powder (8 g1\
The medium, glassware and instruments were autoclaved at 121 °C for 20 min. In all embryo germination experiments, the seeds were decontaminated for 15 min in 3.5% sodium hypochlorite, and then rinsed three times in sterile distilled water.2.2.3.1 Decontamination of explants
To compare decontamination procedures, the stored seeds of Mariya and Oniya clones were decontaminated after they were soaked in distilled water for 30 min (water pretreatment) or without prior soaking in water (without water pretreatment).
Decontamination was done for 15 min in 3.5% sodium hypochlorite then rinsed three times in sterile distilled water. Embryos were then excised and inoculated onto MS medium without plant growth regulators. A 2 x 2factorial experiment was carried out in a completely randomized design (CRD). Thirty test tubes per treatment in two replications with one embryo per test tube were used. Further studies on methods of decontamination of seeds and/or embryos were carried out using seeds of wild enset type W02. In this case, seeds were first decontaminated in 3.5% sodium hypochlorite for 30 min and rinsed three times with sterile distilled water. Then embryos were excised and divided into two groups, each group consisting of24 embryos. Embryos of the first group were inoculated onto medium without further decontamination, while embryos in the second group were decontaminated in 3.5% sodium hypochlorite for 5 min, rinsed three times with sterile distilled water and then inoculated onto the medium. All embryos were placed horizontally on MS medium containing 0.5 mg
r
1 BA + 0.2 mgr
1 IAA and 5g1"1 AC.
2.2.3.2 In vitro embryo germination
For experiments on in vitro germination of embryos from stored seeds of Mariya and Oniya genotypes, factorial combinations of two types of embryo orientations on the three different medium compositions were used in a CRO. Embryo orientations on the medium were vertical (haustorium embedded in medium with the meristematic region exposed) or horizontal (longitudinal axis of the embryo was placed flat halfway embedded into the medium). The composition of the media were: MS without plant growth regulators (PGRs) and MS supplemented with (mg
r
1) 0.5 BA + 0.2 IAA or 0.5 BA + 0.2 2,4-0. After inoculation, the cultured embryos were transferred to a growth room and incubated in the dark at24 QC. Seedlings were then transferred to irradiances of 4-6 Ilmol m·2S·1 for a week and thereafter to 43 Ilmol m·2S·1. In vitro germination of embryos from seeds of two wild enset types (W01 and W02) was investigated on six media compositions with and without activated charcoal (AC) in factorial treatment combinations. After seed decontamination, the embryos were aseptically excised and inoculated horizontally onto the medium. The medium compositions were: MS without PGRs and MS supplemented with (mgr
1) 0.5 BA + 0.2 IAA; 0.5 BA+ 0.2 2,4-0; 0.5 BA + 0.2 IAA+ 0.2 2,4-0; 1.5 BA + 2 2,4-0 or 1.5 BA + 2 2,4-0 + 0.2 IAA. Based on the treatments, 5 g
r
1AC was used. Twenty-four embryos per treatment, one embryo per test tube in two replications, were used. Explanted embryos were incubated in a growth room with a 16 h IighU8 h dark and irradiances of 43 Ilmol m-2S-l.2.2.4 Data collection and statistical analysis
Data on germination and contamination, number of shoots per embryo, shoot and root length and number of roots and leaves per shoot were collected. Percentage of explants without blackening was used to assess the effect of AC on blackening of cultured embryos. Callus formation was considered when explanted embryos produced clearly observable callus usually at the base of the shoots but sometimes embryos formed callus without producing radicles and epicotyls, which was used as indications of germination. Percentages of callus formation and of explants without blackening were computed based on growing embryos. Embryos were considered growing when they gave rise to shoots and/or callus. GenStat 5 Release 4.2 (McCONWAY et al. 1999) was used to analyze the data. Significant means were separated by least significant differences (LSD) at a 5% probability.
Standard errors of means (SE) were also computed. A Correlation matrix was run to explain associations of growth parameters.