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Sterilization procedure
The same batch of seeds used in the preliminary trials was used. Firstly, the hairs on the seeds were removed by hand, in order to better expose the seed coat to sterilants during surface sterilization (Figure 2.4A, Figure 2.4B). Each seed was then carefully screened by hand, and only plump, healthy-looking seeds were used. The seeds were surface-sterilized in 0.35% sodium hypochlorite (NaOCl) for five minutes, followed by three minutes in concentrated sulphuric acid (H2SO4). Thereafter, the seeds were stirred in sterilized distilled water for 10 minutes. Each seed coat was then cut open using a scalpel and a pair of forceps, and the embryo removed (Figure 2.4C). The embryos were carefully placed into the medium, in an upright position.
Medium treatments
Three different types of media were used in this experiment, which all contained halfstrength MS medium (Appendix A) as the basal medium. Forty explants were used in each treatment. The first medium contained no growth regulators, the second contained 1 mg l-1 GA3, and the third medium contained 10 mg l-1 GA3. All the media were also supplemented with sucrose (3%), and Gelrite® (3 g l-1). The pH of all three media was adjusted to 5.7 before autoclaving. Ten ml of each medium were dispensed into 50 cm3 glass test tubes, and capped. The media were sterilized in an autoclave (Hirayma Hiclave®, Model HA – 300D) at 104 KPa at 121ºC for 20 minutes.
Growth condition treatments
Excised zygotic embryos were germinated in either light or dark conditions. For the light treatment, a 12-hour photoperiod was used. Cool, white fluorescent tubes provided 60 μmol m-2 s-1 Photosynthetic Active Radiation (PAR) to the explants. In the other treatment, the explants were grown in total darkness for the entire germination period.
Two different temperature regimes were used to study the effects of temperature on the germination of the embryos exposed to the two light regimes. For the first temperature treatment the embryos were kept in a growth chamber at 25±2ºC for the entire germination period. In the other treatment, alternating temperatures of 21±2ºC/12±2ºC (light/dark) were used. The embryos were considered germinated when the growth of the radicle took place.
Transfer to ex vitro environments
Twenty in vitro-germinated embryos were planted out either into pasteurized silica sand or into a mixture of peat, coconut coir and sand (1:1:1 v:v:v), with no bottomheating. After removing the plantlets from the test tubes, they were carefully washed in tap water to remove any medium attached to them. The plantlets were then transplanted to a fibre-glass-covered mistbed facility equipped with a fogging system, which kept the relative humidity above 95%, and a water sprinkler system that irrigated every 2 minutes for 30 seconds. The Photosynthetic Active Radiation (PAR) of the mistbed was 400 μmol m-2 s-1, and the average temperature was 28±2ºC.
Statistical analysis
A completely randomized design was used in all the experiments. Significant differences in the germination percentage between treatments were tested using Chisquare analysis. Means of root and cotyledon fresh mass were separated using Tukey’s studentised test at the 5% level of significance. All statistical analyses were performed using the SAS program (SAS Institute Inc, 1996). ANOVA is shown in Tables C1 and C2, Appendix C.
CHAPTER 1 LITERATURE REVIEW
1.1 Introduction
1.2 In vitro propagation
1.3 References
CHAPTER 2 IN VITRO CULTURE OF ZYGOTIC EMBRYOS OF PROTEA CYNAROIDES AND ITS ESTABLISHMENT IN EX VITRO ENVIRONMENT
2.1Abstract
2.2 Introduction
2.3 Preliminary trials
2.4 Main trial
CHAPTER 3 MICROGRAFTING OF PROTEA CYNAROIDES
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.4 Results
3.5 Discussion
3.6 Conclusion
3.7References
CHAPTER 4 PLANTLET REGENERATION OF PROTEA CYNAROIDES THROUGH DIRECT SOMATIC EMBRYOGENESIS AND MULTIPLE SHOOT DEVELOPMENT
4.1 Abstract
4.2 Introduction
4.3 Materials and methods
4.4Results
4.5 Discussion
4.6 Conclusion
4.7References
CHAPTER 5 ROLES OF STARCH AND PHENOLIC COMPOUNDS IN VEGETATIVE PROPAGATION OF PROTEA CYNAROIDES STEM CUTTINGS
5.1 Abstract
5.2 Introduction
5.3 Materials and methods
5.4 Results
5.5 Discussion
5.6 Conclusion
5.7 References
CHAPTER 6 ALLELOPATHIC ACTIVITY OF PHENOLICS FROM PROTEA CYNAROIDES STEMS, AND THEIR ROLE AS ENDOGENOUS ROOTING REGULATORS IN VIVO
6.1Abstract
6.2 Introduction
6.3 Materials and methods
6.4 Results
6.5 Discussion
6.6 Conclusion
6.7 References
CHAPTER 7 GENERAL DISCUSSION AND CONCLUSION
7.1 References
SUMMARY
