The large geophytic monocotyledon, Hypoxis colchicifolia Baker, has been identified for the importance of its corm extracts in the development of a potential non-toxic prodrug for the treatment of inflammation, certain malignancies and HIV-infection. The underground corms of this plant are also commonly used for therapeutic applications in traditional medicine in Kwazulu-Natal where it primarily occurs. A review of published literature revealed, however, that H. colchicifolia plants are currently harvested in an unsustainable manner from traditional collecting sites due largely to population growth, increased land use for urban development and agriculture, and the popularisation of Hypoxis plants for herbal remedies. A further search of historical records established that H. colchicifolia plants were dominant in grassland vegetation prior to 1950, but had rapidly declined since then. Quantitative data subsequently gathered in this study from comparative surveys of both H. colchicifolia and H. hemerocallidea populations from sites with near-pristine, disturbed, burnt and mown grassland vegetation showed for the first time that exposure to human activity and the grassland management practices of mowing and burning incurred not only a 75% reduction in plant density of both these Hypoxis species, but also the total destruction of mature plants of H. colchicifolia in frequently mown and burnt areas. Flowering data recorded in these surveys, and confirmed by monitoring field performance of cultivated H. colchicifolia plants, showed that a contributing factor to the plant's inability to withstand these pressures was that juvenile forms only reached flowering maturity after three to four years growth, thus adversely affecting seedling recruitment. It was concluded therefore that, since Hypoxis species responded differently to mowing and burning, geophytic plants should be considered individually and not as "forbs" during the planning of grassland management programmes for natural conservation areas. The need to cultivate H. colchicifolia to ensure its survival was also established using the new field data gathered in this study. Methods to propagate this species have, however, not been established. Data gathered on all the plants comprising a single population confirmed that mature plants survive to an estimated 20 years and longer in natural areas. Greatest hypoxoside yields were also obtained from corms with a fresh mass of 350g to 400g. Since these corms were estimated to be 10-years-old and older, propagation and cultivation methods that could sustain plant production and survival for long periods, and therefore increased hypoxoside yields, would have to be developed. Several micropropagation systems suitable for the mass production of H. colchicifolia and from which phenotypically normal plantlets were recovered, were therefore established via organogenesis, embryo culture and somatic embryogenesis. The latter cultures have not been reported previously for Hypoxis. In the former culture the toxic effects of phenolic leachates and browning were controlled, and improved plantlet regeneration achieved, by adding polyvinyl pyrrolidone to the medium and introducing distinct sequential aseptic steps into the micropropagation procedure developed. Defined protocols for the different phases of in vitro somatic embryogenesis are not readily available for monocotyledons, however, neither are the factors controlling embryogenesis and organ regeneration known. In this study the process of somatic embryogenesis from excised zygotic embryos of H. colchicifolia was shown to be complex and the resultant cultures very heterogeneous. Although the stage of development of the zygotic embryo explants was important at the time of inoculation, data showed that the induction and regulation of the processes of embryo culture and somatic embryogenesis were ultimately determined by the exogenously applied plant growth regulators. By comparing the different pathways leading to plantlet regeneration, and the morphological stages of development of the structures produced both on solid and in liquid media, not only photographically, but also quantitatively and schematically, the repeated formation of pseudoembryonic structures and neomorphs confirmed that they form an integral part in the in vitro somatic embryogenic pathway of H. colchicifolia. Evidence suggested not only that two types of somatic embryos are produced in the embryogenic cultures of H. colchicifolia, but that the pseudoembryonic structures produced resemble the pseudobulbils produced in polyembryonic cultures of Citrus. The success of the somatic embryogenic cultures was confirmed by the estimation that 28 112 somatic embryos and embryo clusters of H. colchicifolia could be obtained from 16 ml of somatic embryogenic liquid culture. Furthermore phenotypically normal plantlets regenerated from all of the micropropagation procedures developed were successfully transplanted from the laboratory, acclimatized under greenhouse conditions and their horticultural and field performances evaluated. / Thesis (Ph.D.)-University of KwaZulu- Natal, Pietermaritzburg, 2004.

Boophone disticha (L.f.) Herb is one of the most widely distributed bulbous species in southern Africa. Of Africa’s many bulbous plants, it is widely known for its poisonous and medicinal properties. It is of considerable ethnobotanical interest in traditional medicine because of its hallucinogenic alkaloids and it has great potential as an ornamental due to its fan-shaped foliage and large umbel of bright pink to deep red flowers. In South Africa, many bulbous plants are used in traditional medicine which are collected from wild populations. The high demand for trade and use of such plants, that are destructively harvested, places an enormous pressure on natural populations. According to the Red List of South African Plants, the conservation status of B. disticha has been listed as ‘declining’. It is, therefore, important to develop conservation strategies for these medicinal plants, such as the development of alternative propagation methods. Micropropagation is a useful technique for rapid clonal multiplication of plant material which could alleviate the pressure on the wild plant populations, as well as potentially producing useful secondary metabolites. The in vitro induction of storage organs is especially beneficial as it can limit the loss of plants during acclimatization since bulblets are generally hardier than shoots or plantlets. Thus, the main aim of this research was to establish a micropropagation protocol which could be a valuable tool for conservation of this plant species. In addition, B. disticha plants were assessed in various ethnopharmacological assays to evaluate their medicinal properties, and a preliminary study on the population genetics was also conducted. As part of the development of a suitable micropropagation protocol, the effect of environmental and physiological factors on the initiation and growth of bulblets were investigated. These factors included the effect of various plant growth regulators, carbohydrates, temperature, photoperiod and liquid culture. Different explants (i.e. ovaries, anthers, filaments, pedicels, embryos, seeds and bulb twin-scales) were tested to determine which explants were the most suitable for subsequent experiments. Although success was limited, twin-scales proved to be the most suitable explant and it was demonstrated that activated charcoal, ascorbic acid and N6- benzyladenine were required as media supplements. Antimicrobial activity was tested between different plant parts and seasons. The plant parts (roots, leaves, outer and inner bulb scales) were extracted with a range of differing polarity solvents. These were screened for antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae, and for antifungal activity against Candida albicans. Extracts from roots of plants collected in spring and summer showed the best antimicrobial activity against B. subtilis, E. coli and K. pneumoniae, indicating that plant part and collection time do affect activity. In vitro grown bulblets also showed antimicrobial activity, demonstrating that antibacterial properties were maintained in cultured plantlets. Extracts from plants collected in summer were tested for mutagenicity using the Ames test (Salmonella/microsome assay; plate incorporation method, with or without metabolic activation). None of the extracts tested were found to induce mutations and also did not modify the effect of the mutagenic compounds (2AA with S9 and 4NQO without S9). Although the results do not indicate a mutagenic response, this does not necessarily confirm that it is not mutagenic nor carcinogenic to other bacterial strains, however, B. disticha must be used with caution, especially considering the levels of alkaloids in the plant. The two major constituent alkaloids of B. disticha were identified as buphanidrine and distichamine. In the antibacterial assay, both compounds exhibited broad-spectrum micromolarlevel activity against the two Gram-positive and two Gram-negative bacteria tested. The best MIC value, of 0.063 mg/ml, was found for bupanidrine/distichamine against S. aureus, E. coli and K. pneumonia. The isolated compounds were tested and found to be neither mutagenic nor toxic at the concentrations tested. Thus, buphanidrine and distichamine are thought to be the constituents likely responsible for the medicinal properties of the plant. To determine the level of genetic variation between different populations of B. disticha, plants were collected from six wild populations in KwaZulu-Natal, South Africa. DNA was isolated and tested for genetic variation using ten Inter Simple Sequence Repeat (ISSR) primers. The level of inter-population polymorphism ranged between 23% and 39%, showing that the populations had low genetic polymorphism. From the genetic distance results, it was found that the Midmar and Umgeni Valley populations are closely related, and these populations are similar to two sister populations. The Amatikulu and Lions River populations were similar but slightly different to the other populations. Antimicrobial assays showed minor difference in activity from the six wild populations. Although the micropropagation of B. disticha had limited success, this study did develop a successful decontamination protocol as well as determine the most useful explant and supplements. This information provides an important starting point for the development of a successful micropropagation protocol for the conservation of B. disticha. Since, B. disticha is an important medicinal plant in South Africa, this study has also deepened our understanding of the constituents that could be responsible for the medicinal properties of B. disticha and, in so doing, confirmed the value of this plant for use in traditional medicine in South Africa. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Cussonia species (commonly known as Cabbage trees) are indigenous to South Africa and are used in traditional medicine to treat an assortment of diseases. Due to their attractive growth form, they are assets in gardens. However, there are no developed methods for propagating these species. The use of three selected species, Cussonia paniculata (Eckl. & Zeyh.), C. spicata (Thunb.) and Schefflera umbellifera (Sond.) Baill, = C. umbellifera), in traditional medicine was validated. Rapid propagation protocols for C. paniculata and C. spicata were investigated and ultimately developed for the former species. Cussonia paniculata, C. spicata and C. umbellifera were screened for their medicinal properties, mainly focussing on anti-bacterial, anti-inflammatory and anti-malarial activities. In the anti-bacterial screening, C. spicata bark and root extracts showed activity against selected Gram-positive and Gram-negative bacterial strains at a concentration of 50 mg ml ¯¹ . The highest inhibition was observed with ethanol and ethyl acetate root extracts against Staphylococcus aureus. The other two species did not show anti-bacterial activity. Ethanol and ethyl acetate extracts of all species showed anti-inflammatory activity in the cyclooxygenase assay (COX-1) at a concentration of 8 μg ml ¯¹, These active extracts showed an inhibition percentage that was greater than 50 % against cyclooxygenase. In the anti-malarial screening , bark extracts were screened. C. umbellifera bark extracts exhibited the best inhibition against P. falciparum, a malaria-causing agent in humans. The percentage inhibition of these extracts was up to 100% at a concentration of 200 μg ml ¯¹ . While C. spicata is known to be used to treat malaria, the screening results showed much less activity (less than or equal to 35 %) as compared to C. umbellifera, which is preferably used to treat malaria. The results obtained from screening these three species validated their use in traditional medicine. This means that the people or traditional healers use these species for different treatments by possibly relying on past knowledge about the effects after administering the medicine. Fingerprinting using Thin Layer Chromatography (TLC) was used in an attempt to determine whether there are any chemical differences or similarities between the three species. There were similarities between the plant parts across the species as well as some differences. However, this method cannot be used as an unequivocal test to deduce that compounds that are present in a certain species and not in others are the ones responsible for bringing about a certain biological activity. That can only be achieved by a bioassay-guided isolation of possible compounds. A tissue culture protocol was developed to produce a large -number of plants of C. paniculata. Explants were derived from nodal explants of in vitro germinated seeds and cultured on Murashige and Skoog (MS) (1962) medium supplemented with 3% sucrose, 2.5 mg l ¯¹ BA and solidified with 3 g l ¯¹ Gelrite. These explants produced multiple shoots. The average number of shoots per explant ranged between 1 to 3.5. Multishoots were subcultured on to rooting media and roots were produced on MS with 0.75 mg l ¯¹ IBA and 1 mg l ¯¹ NAA. Callus from zygotic embryos also produced plantlets on MS supplemented with 1.5 mg l ¯¹ 2,4-D and 0.5 mg l ¯¹ BA. Hyperhydricity was encountered in this study. This problem was reversed successfully by transferring the shoots from medium solidified with 3 g l ¯¹ Gelrite to medium solidified with 8 g l ¯¹ agar. Plantlets were successfully acclimatized for planting ex vitro. The percentage of healthy plants after a 35-day acclimatization period was 63 %. C. spicata was not successfully micropropagated from shoot-tip explants. However, a protocol was developed for decontaminating shoot-tips from the mother plants. The plant material was successfully decontaminated with 0.01% HgCl₂ for 15 min. The decontamination percentage was up to 80 %. Browning of the explants was observed and it was successfully treated with soaking the explants in a 15 mg l ¯¹ ascorbic acid solution for 15 min. A high percentage of shoot-tip regeneration (80 %) was observed when they were cultured on MS medium supplemented with 2 mg l ¯¹ BA, 1 mg l ¯¹ IAA and 1 mg l ¯¹ GA₃. However, multishoots were not observed as in C. panicualata. Shoot elongation in vitro was similar to shoot elongation as it occurs in nature. The shoots elongated and a flush of palmitately arranged leaves were produced. Further research is required to investigate a commercially viable protocol for rapid propagation and conservation of the germplasm of Cussonia species. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Hybrid clones of the fast-growing Eucalyptus grandis and cold-tolerant E. nitens (GN clones) have been identified by the South African Forestry Industry as being highly suitable for plantations in cold-dry marginal areas. However, one of the main problems regarding their propagation is the difficulty in rooting of cuttings, both in vitro and ex vitro. The aims of this investigation, therefore, were (1) to develop widely applicable and efficient in vitro rooting system(s) for these commercially important clones, and (2) to assess some physiological characteristics of the roots produced. Adventitious shoots (15-20 mm in length) were obtained (l0 shoots/explant) from axillary buds on Murashige and Skoog's (MS) medium containing 0.01 mg.l-1 NAA, 0.01 mg.l-1 IBA and 0.2 g.l-1 FAP. The effect of various medium components, as well as modification of culture environment on in vitro rooting, were investigated. The highest rooting frequencies in clones GN121 (75%) and GN107 (65%) were achieved on l/4 MS with additional 0.22 g.l-1 CaCl2..2H2O and 0.18 g.1-1 MgS04.7H2O, 0.1 mg.l-1 IBA, 0.1 mg.l-1 biotin, 0.1 mg.l-1 calcium pantothenate, 15 g.1-1 sucrose and 4 g.l-1 Gelrite. Best culture conditions were an initial 72-hours dark incubation followed by a 16-hours day/8-hours night photoperiod at a PPFD of 37 µmol.m-2.s-1 and 23°C day/21°C night for seven days, after which the PPFD was increased to 66 µmol.m-2.s-1 at 27°C day/21°C night for 18 days. Towards the development of a more widely applicable in vitro rooting protocol for GN clones, the use of Agrobacterium rhizogenes strains was investigated. Production of transgenic roots was observed on carrot discs and shoots from seedlings of Eucalyptus grandis and E. nitens, but not on shoots of GN clones. Therefore, a method needs to be established for the successful transfer and integration of the Ri plasmid of Agrobacterium into the hybrid plant genome for induction of transgenic roots. The quality of roots produced in vitro and from cuttings was assessed by examination of root anatomy and hydraulic characteristics. Adventitious roots were prepared for measurement of hydraulic conductivity by detopping explants, then filtered, acidified distilled water was drawn through undisturbed potted root systems under partial vacuum, causing no damage to the roots. Initial studies showed that tissue culture-derived roots exhibited a higher specific root mass hydraulic conductivity than those derived from cuttings (6.46 x 10-6 vs. 3.06 X 10-6 g.kPa-1.s-1.g-1 dry root), probably due to root architecture. Curves relating vulnerability to water potential were constructed and both types of roots showed vulnerability to cavitation at high water potentials. Differences were also observed in staining reactions (safranin and fastgreen) which might suggest differences in presence and level of secondary metabolites in these roots at the juvenile stage. Applications of the developed protocols and future research strategies are discussed. / Thesis (M.Sc.)-University of Natal, Durban, 1999.

Recalcitrant seeds are those that are shed at high water contents, are actively metabolic throughout development, when they are, and remain, desiccation-sensitive, and may also be chilling sensitive. These properties preclude their conventional storage. Because recalcitrant seeds lose viability rapidly (within a few days to several months depending on the species) the long-term storage of their germplasm is achievable only by cryopreservation [i.e. storage at very low temperatures, generally in or over, liquid nitrogen at -196°C or -150°C, respectively. Generally the seeds are far too large to be cryostored, thus explants - most conveniently, excised zygotic embryonic axes - are used. As the axes of recalcitrant seeds are highly hydrated, specific pre-treatments prior to freezing have to be applied in order to avoid lethal ice crystal formation. During the course of this study, cryopreservation protocols were developed for excised zygotic embryonic axes of two different species (Quercus robur L. and Ekebergia capensis Sparrm.). Surface-sterilisation regimes were tested for axes of both species, with the use of a 1% sodium hypochlorite solution containing a wetting agent, emerging as the best. For both species, the vigour and viability of axes, assessed by in vitro germination performance, was tested after the implementation of four different rates of desiccation (achieved by a laminar-airflow; silica-gel-; flash- and fast flash-drying). The most rapid dehydration rate (fast flash-drying) facilitated the best germination rates (vigour) for both Q. robur and E. capensis axes after 240 and 20 min, when water contents were reduced to 0.37 ±0.04 and 0.39 ±0.06 g g-1 (dmb), respectively. Consequently, fast flash-drying was used in combination with three different freezing rates (slow, intermediate and ultra-rapid cooling). While axis viability was lost after slow or intermediate cooling, good survival was obtained for each species after ultra-rapid cooling. In addition to the optimisation of culture conditions, desiccation and freezing rates, the efficacy of different thawing media (distilled water, mannitol, sucrose, full-strength MS medium supplemented with sucrose and a 1 µM calcium/1 mM magnesium solution) was also assessed. The only thawing medium that ensured normal seedling production was the Ca2+Mg2+ solution, in which electrolyte leakage was significantly curtailed. In addition to vigour and viability assessment the responses of the embryos to the various manipulations were monitored by light microscopy and/or transmission electron microscopy. The results of the various manipulations are discussed in terms of the stresses imposed on the excised axes, by each of the procedures. For axes of Q. robu, the outcome of the presently developed successful procedure and two unsuccessful protocols from the published literature are compared and contrasted. It is concluded that while in vitro germination media need to be assessed on a species basis, use of the mildest effective surface-sterilant, in conjunction with the most rapid means to achieve dehydration and cooling/freezing, are likely to underlie generally successful cryopreservation. Additionally, thawing parameters have emerged as being critically important. / Thesis (M.Sc.)-University of Natal, Durban, 2000.

In South Africa, Eucalyptus trees are used for many processed wood products (e.g. paper) and in the mining industry. Priorities in Eucalyptus breeding programmes include selection of varieties that are fast growers, insect and disease resistant, have appropriate wood characteristics and can grow in a wide variety of environmental conditions. Cold-tolerant cultivars of E. saligna and of E. grandis have been bred and selected in Australia and South Africa, respectively for use in cold regions of Natal Midlands and North Eastern Cape. However, the production of large numbers of such cultivars for planting out in a commercial scale is being impaired by slow growth rate, low regeneration time and poor rooting ability of cuttings from these trees. Consequently, methods of in vitro propagation of cold-tolerant clones were investigated. Axillary buds were induced and subjected to a variety of multiplication, elongation and rooting media. The optimised protocol for the production of shoots from axillary buds was: bud induction medium comprising of MS supplemented with 20 grl sucrose and 10 grl agar for 1-2 weeks, multiplication medium comprising of MS supplemented with 0.1 mgrl biotin, 0.1 mgrl calcium pantothenate, 0.2 mgrl benzyladenine phosphate, 20 grl sucrose and 3.5 grl Gelrite for 4 weeks, elongation medium for 4-6 weeks comprising of MS medium supplemented with 0.1 mgrl biotin, 0.1 mgrl calcium pantothenate, 0.35 mgr' NAA, 0.1 mgr' kinetin, 0.1 mgrl IBA, 20 grl sucrose and 3.5 gr1 Gelrite. Production of plantlets via somatic embryogenesis was also investigated but hampered because of high rates of contamination as pieces of mature leaves were used as exp1ants. Ongoing breeding programmes are aimed at obtaining hybrids of Eucalyptus that are cold tolerant. The hybrid progeny then need to be screened for cold-tolerance. However, a major problem in the selection of cold-tolerant clones is that diagnosis can only be undertaken by assessing the field performance of the genotypes under various environmental conditions. In this regard, a protocol for 1D gel electrophoresis was developed for Eucalyptus species with the view to use it for the detection of cold-tolerant stress proteins. Leaf material from both non-cold tolerant and cold-tolerant clones was used. Well-resolved gels that focused on the comparison' between protein profiles of cold-susceptible and cold-tolerant clones before and after period of cold stress were obtained. The findings of this study showed that two polypeptides, one in the lower molecular region of 14.3-20.1kD and another of a higher molecular weight in the region of 116.4-170 kD were observed after cold acclimation. These changes in polypeptide profiles were observed in cold-tolerant E. grandis x nitens (GN1) and E. saligna (AS 184, AS 196 and TS 15) but not in a non-cold tolerant species E. grandis (TAG 731). These polypeptides may have an important role in the cellular adaptation to cold temperatures. It is suggested that this method may be used as a diagnostic tool for screening cold tolerance on Eucalyptus cultivars. / Thesis (M.Sc.)-University of Natal, Durban, 1996.

Cereal crops such as maize and sorghum are economically important in South Africa (SA) as a staple food diet. In order to meet the needs of South Africa’s growing population, higher yields in crop production need to be attained. However, the two major stress factors that affect yield production and require primary attention are nutrient deficiencies and pest infestations. Research is now being focused on certain endophytes that have become a valuable tool for agriculture as they protect crops against the above-mentioned stresses. The endophyte focused on in this study was Arbuscular Mycorrhizal fungi (AMF). This research was aimed at developing an in vitro culture system for SA cereal crops to enable interaction studies of endophytes. This dissertation is divided into two parts; the first part focused on the development of an in vitro culture system, the assessment of sorghum plant growth and exudate production in the presence of the Glomus intraradices strain. The results indicated that sorghum produces the required root exudates in the second stage of growth. Using high pressure liquid chromatography with mass spectrometry (HPLC/MS), it was noted that sorghum produced phytochemicals as chemoattractants for the respective endophytes. However, it was documented that when the plant underwent certain stresses they produced exudates, which acted as phytotoxic compounds that destroyed symbiotic organisms around sorghum rhizophere. The second part focused on optimization of the surface sterilization of maize seeds. The results indicated that maize contained unidentified endophytes, which negatively affected plant development. Surface sterilization of maize seeds was accomplished. The successful in vitro development can be used for future use to study plant development. Understanding plant development and interaction with symbiotic endophytes would not only be of great benefit but would also make it easier to create a biocontrol agent in vitro, which would bring about high crop yields at cost-effective prices and would be less labour intensive. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Coconut (Cocos nucifera L.) is native to the regions between 20oN and 20oS of the Equator, where it plays a significant socioeconomic role in the local communities. There it is referred to as ’The Tree of Life’, a eulogistic epithet describing its versatile use - more than 100 edible and non-edible products can be produced from it. Therefore the coconut palm is grown in about 90 tropical countries on more than 10 millions ha of land (Hamon et al., 1999). Although coconut has a high local socioeconomic reputation, its production is experiencing many problems and consequently the area planted with this crop is declining. The conventional breeding approach using seed to replant land is very expensive due to the low production of seed for planting, and even when elite germplasm is available it takes decades to multiply up enough planting material for new areas (Adkins et al., 1999). Hence over the past 40 years research has been directed towards developing a new technique for the micropropagation of coconut using somatic embryogenic approach. Throughout this time however one conclusion is repeatedly made – coconut is very recalcitrant to somatic embryogenesis. And although the many obstacles to this are slowly being reduced, in order to successfully micropropagate coconut on a large scale bottlenecks in the protocol still exist, and those include inconsistency of the embryogenic response by explanted tissues, poor somatic embryo maturation and germination, low regeneration rate of the new plantlets and long time required to produce plants (1.5 years) (Samosir et al., 1998). These bottlenecks and other problems were researched in the present study with the aim of trying to speed up the efficiency of coconut somatic embryogenesis process. Hence this thesis had the objectives to identify a starting protocol for coconut somatic embryogenesis; to select an appropriate for aim that explant; to optimize the production of embryogenic callus; to increase the rate of initiating coconut somatic embryos; to improve the maturation of somatic embryos and their germination efficiency; and to optimize the regeneration rate of the new plantlets. In order to identify a starting protocol, preliminary work was conducted, where existing protocols for coconut somatic embryogenesis were compared in their efficiency to induce somatic embryos. The protocol that stood out as the best in producing most embryogenic callus and subsequently embryos, as well as having the least dead (in culture) explants, was that of Nikmatullah (2001). Therefore the latter was chosen to be used as a starting protocol for this study. New sources of explants were investigated during the current work as well, using tissues from different parts of in vitro derived 8 months old coconut plantlets. Those however have shown to be unsuitable for somatic embryogenesis, since only non-embryogenic callus was developed by some of the inoculated tissues. The immature inflorescence explants were superior in producing embryogenic callus and somatic embryos; therefore they were selected as the preferred explant source to use in the next steps of the current study. Optimizing the production of embryogenic callus was the first issue to address during the core work of this project. As a result of that the culture conditions were considerably improved by using vessels with larger headspace-medium ratio (3:1), as well as by selecting younger immature inflorescences and transversely segmenting the top half of the inflorescence spikes into smaller size (1 mm) sections. Further improvement was possible by studying the make up of the callus growth media. Amongst the administered for that purpose substances the applied together polyamines spermine (0.10 µM) and putrescine (7.5 mM) have proven to play a notably positive role in the induction of callus from coconut immature inflorescence explants. Thidiazuron (TDZ, 10 µM) too has shown a potential to improve the efficiency of the initial stage of coconut somatic embryogenesis, but only when applied in conjunction with other cytokinins (eg. BAP and 2iP). Smoke-saturated-water (SSW, 10 %) could only slightly diminish the amount of necrotising cultured explants, and high 2,4-D concentrations could not support the induction of callus from immature coconut inflorescences. Collectively taken, as a result of this current study the production of callus was improved by 300 %. The rate of coconut somatic embryos formation was as well significantly increased (over 300 %), by the simultaneous application of suspension culture step, spermine (0.01 µM), SSW (10 %) and high auxin concentration (500 µM). Nevertheless the presence of TDZ and other cytokinins in the medium, as well as the absence of activated charcoal, were found to be unable to positively influence the somatic embryogenesis process. Despite the considerable improvements made in the efficiency of inducing callus and initiating embryos, the poor maturation and germination (eg. 5 %, Verdeil et. al., 1999) of somatic embryos still remained a bottleneck to the whole somatic embryogenesis procedure. Therefore further work was conducted in that direction and discovered that embryo maturation and germination rate can be elevated to 55 % by administering ancymidol (30 µM) to the somatic embryo maturation medium. This plant retardant has exhibited here three potential modes of action towards the cultured coconut somatic embryos: a) as a promoter of somatic embryo maturation and germination; b) as a preventor of pre-germination death of the somatic embryos; and c) as a preserver of non-germinating somatic embryos, that still can possess the potential to germinate in the future. The work during the next step of the process – regeneration of the new plantlets – has shown that the omission of plant growth regulators from the media was crucial for the development of germinated embryos into new plantlets, where otherwise no plant regeneration occurred at all. The achieved here plantlet regeneration rate in the PGR-free medim was 56 %, which is higher than the previously reported 20 % regeneration rate (Verdeil et al., 1994) for coconut plantlets produced from immature inflorescences explants. As a result of this current work a new method was developed for somatic embryogenesis of coconut from immature inflorescences explants (Fig. 9.2). The overall efficiency of this protocol is over three times higher than that of the starting protocol (Nikmatullah, 2001) selected during the preliminary work. Furthermore, when using this new method the entire duration for regenerating clonal coconut plantlets (up to the stage of first root and shoot emerging) takes up to 8 months, which is the shortest reported time for producing coconut plantlets via somatic embryogenesis (eg. 36 months from inflorescences explants (Verdeil et. al., 1999) and 18 months from sliced zygotic explants (Samosir, 1999, Fig. 9.2), presenting an additional valuable advantage of this newly developed method, from the perspective of the potential to micropropagate coconut on a commercial scale.

Diss. (sammanfattning). Alnarp : Sveriges lantbruksuniversitet, 2005. / Härtill 4 uppsatser.