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Please use this identifier to cite or link to this item: http://hdl.handle.net/123456789/2864

Advisors: Professor Legesse Negash
Keywords: Ethnobotany Ethiopia
medicinal plants,
seed germination
in vitro regeneration
Copyright: Jan-2009
Date Added: 10-May-2012
Publisher: AAU
Abstract: Studies on the phenology of Plumbago zeylanica L.(Plumbaginaceae), and propagation biology of other four highly threatened medicinal plants, namely Echinops kebericho Mesfin (Astraceae), Glinus lotoides L. (Mol luginaceae) Securidaca longepedunculata Fres. (Polygalaceae) and Taverniera abyssinica L. (Leguminosae) were conducted with a view to conserving and developing these threatened medicinal plants. The objectives of this research were (1) to study the reproductive biology of P. zeylanica; and (2) to develop (i) seed-based propagation methods of E. kebericho, G. lotoides and S. longepedunculata, (ii) vegetative propagation by stem cuttings of G. lotoides, and (iii) in vitro regeneration of T. abyssinica. Mature seeds were used as the starting plant material for all the studied species. Different parameters affecting seed germination and seedling establishment were studied for E. kebericho, G. lotoides and S. longepedunculata. Seedlings were only used for T. abyssinica and G. lotoides in tissue culture and vegetative propagation techniques, respectively. Studies on the phenology of P. zeylanica were conducted under glasshouse and nursery conditions using several parameters such as plant size, seasonal climate, and hormone application and mating system. A tissue culture protocol was tested from several explants, on two basic media and with hormone treatments for T. abyssinica. Seeds of E. kebericho sterilized for 9 and 5 minutes in 70% ethanol and in 10% sodium hypochlorite, respectively, g erminated best (95.2 ± 1.2%) on Murashige and Skoog medium, supplemented with 10 g l_1 phytoagar. Further incre ases or decreases in sterilization time decreased percentage germination and increased contamination, respectively. Untreated seeds (control) were completely contaminated before the emergence of radicle as a result of fungi growth. Seed germination percentage declined with increasing storage time and dropped from 95 ± 0.4 % to 32.2 ± 1.2% in 15 months. Twenty-five degrees centigrade was an optimal temperature for best germination (94.6 ± 2.4%) of seeds compared to others. Seeds sown in pots containing a mixture of sand, nursery soil, and animal manure in a ratio of 0.5:2.5:0.5 respectively, germinated significantly (p< 0.05) compared to other soil ratios. Increases in sand or animal manure ratios decreased germination, while increases in nursery soil increased percentage and rate of germination. High percentage (96± 0.5%) germination was obtained with the seeds sown on nursery soil–surface mixed additives compared with the control. Seeds stored for less than 5 months, and at 25 oC, were the most suitable for in vitro and ex vitro germination of E. kebericho. Seedlings of nursery bed origin survived best compared xiii to those in vitro or pot origin seedlings. Seeds of G. lotoides treated with water at 70o C for 10 to 30 minutes or GA3 (10-3 and 10-4 M) did not show significant (p<0.05) difference in germination compared with the control. Seeds sown in pots containing a mixture of nursery soil, cattle dung, and sand in a ratio of 2:1:0.5, respectively, germinated best (91.6%) compared to other soil ratios, which showed rapid reduction in germination percentage with increases in cattle dung or sand. Seeds stored for 2 months gave best germination (93.7%) compared to ones stored for 5, 8 and 11 months, which showed decreases with increasing storage time. Apical stem cuttings gave the highest rooting percentages (90.2%), root number (8.02) and root length (6.18 cm) with or without hormone treatment than basal stem cuttings. In general, the number and length of roots decreased with applied indolebutyric acid (IBA) concentration. The highest rooting percentage (98.2%) was obtained in a rooting medium consisting of sand, nursery soil, and cattle dung, in equal proportions followed by 1.5:1:0.5 ratios of the same constituents. The percentage of survived rooted cuttings decreased with increasing age of stockplants from which the cuttings were derived. Rooted cuttings obtained without IBA treatment survived significantly (P<0.05). The study found that G. lotoides can effectively be propagated by both sexual and asexual means provided that germination media of specific are employed, and the apical cuttings derived from young seedlings are used for maximal rooting responses. Seeds of P. zeylanica germinated best and vigorously grown (on a mixture of nursery soil and cattle dung in a ratio of 3:1 filled in pots (glasshouse) or on nursery bed-surface mixed cattle dung) as a prerequisite for vegetative and flowering phenological studies. Hypogeal germination characterizes the emergence of seedlings. Subsequent vegetative and flowering phenology between glasshouse and nursery seedlings showed significant difference (p<0.05) in terms of time, duration and yield. Glasshouse seedlings completed their phenophases (aseasonally) within 105 days while nursery seedlings extended to 225 days after seed sowing. Rainy season was the cause for the continuous damage of apical shoots, and consequently stunted vegetative growth of nursery seedlings. Plant size (≥ 95 cm in height), leaves number (33-38) and seasonal climate (wet season) were found to be the most signals for the initiation of flowering buds. Hundred ppm GA3 was the most effective for early flowering (i.e., before 6 days) and production of higher number of flowers (32.6 ± 1.6%) compared to the control (22.5 ± 1.33%). The mode of reproductive biology appeared to be cross pollination and showed significance (p<0.05) compared to the control. The highest flowering (92.40 ± 0.52%) and/or seed-set (85.23 ± 3.55%) were obtained under glasshouse condition xiv compared to the nursery, which dropped as low as 50% in seed-set due to the damage of apical shoots during rainy and cold seasons, and differences of the adaptation of the species under both conditions. In vitro and ex vitro seed germination, extent of seedling survival and establishment has been developed for S. longepedunculata. Seeds treated with gibberellic acid (10-3 M GA3) germinated best (>94.6+1.32%) on Murashige and Skoog medium supplemented with 8 g 1-1 phytoagar or on the sterilized sand filled in a glass culture vessel. Seedlings germinated on MS survived best (81.6 ± 1.34%) and formed strong stems, multi-roots and many branchlets compared to the sand origin seedlings, which showed a high mortality rate upon transfer to glasshouse conditions due to hypocotyl elongation and a poorly developed root system. Seeds sown in pots containing a mixture of sand, nursery soil, animal manure in a ratio of (2:0.5:0.5, respectively) gave highest germination (94.6 ± 2.14%) compared to other ratios of soil mixture. Percentage germination decreased with decreases and increases of sand and nursery soil ratios, respectively. Only a poor germination (>50%) was obtained with seeds sown on the seedbed. Seeds stored at 25 oC germinated best compared to others and the seed viability was declined with storage time. The best in vitro germination ((96 + 0.6%) of T. abyssinica seeds and vigorous seedlings growth as a prerequisite for the development of tissue culture methods was obtained on Murashige and Skoog medium, supplemented with 12 g 1-1 phytoagar without sucrose. Light green compact calli from node, petiole and shoot meristem explants were efficiently induced on Gamborg medium containing 0.90 or 1.80 μM dichlorophenoxyacetic acid (2,4-D) combined with 2.22 μM 6-benzylaminopurine (BAP), and supplemented with 30 g 1-1 sucrose and 5 g 1-1 phytagel. Callus induction and plant regeneration has been established for dingetegna, Taverniera abyssinica. Light green compact calli from node, petiole and shoot meristem explants were efficiently induced on Gamborg medium containing 0.90 or 1.80 μM dichlorophenoxyacetic acid (2,4-D) combined with 2.22 μM 6-benzylaminopurine (BAP), and supplemented with 30 g 1-1 sucrose and 5 g 1-1 phytagel. Callus initiation from shoot meristems and nodes was faster and occurred with a higher frequency than callus initiation from petiole and leaf segments. A high frequency (100%) of shoot regeneration was obtained upon transfer of calli onto regeneration medium containing 8.88 μM BAP combined with 1.14 μM indoleacetic acid (IAA). Regenerated shoots were transferred to rooting medium, which turned out to be optimal when half strength B5 medium was supplemented with 9.84 μM indolebutyric acid (IBA). Upon transfer to glasshouse, 86% survived and grew vigorously. xv The main results indicate that both in vitro and ex vitro seed-based can be used for the propagation of E. kebericho, S. longepedunculata and G. lotoides although in vitro seed culture of G. lotoides showed less germination even in the absence of contamination. G. lotoides was successfully propagated by stem cuttings without hormone treatments. Rainy season, plant size, leaves number, low temperature, cross pollination and glasshouse conditions were found to be the most determining factors for the phenology of P. zeylanica. Light green compact calli, high frequency of shoot regeneration and regenerated roots of T. abyssinica were successfully obtained and acclimatized upon transfer to glasshouse conditions. However, further studies on the development of tissue culture, genetic analysis and the ecological requirements are the next steps for the effective use of the propagation protocols developed by this study.
URI: http://hdl.handle.net/123456789/2864
Appears in:Thesis - Biology

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