The effect of phytohormones on breaking of dormancy of axillary buds in anchote and their subsequent shoot proliferation were examined. Anchote is an annual trailing endemic plant with high calcium content grown for its edible tuberous roots in Ethiopia. Nodal explants harvested from the greenhouse were sterilized using various concentrations of a commercial bleach (JIK) which contains 3.85% sodium hypochlorite (NaOCl) and time duration. The highest (85%) clean explants were obtained when 5% JIK was used for 15 minutes. The explants were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of benzylaminopurine (BAP), kinetin, and thidiazuron (TDZ). The highest frequency of microshoot induction (84%) and mean number of microshoots (3.4) were recorded from explants cultured on medium supplemented with TDZ 0.025 µm. Hyperhydrated shoots were observed on media supplemented with high concentrations of BAP and kinetin but interestingly not on TDZ media. Induction of roots was highest (86%; 4.6 roots per shoot) when shoots were transferred to half strength MS medium containing 0.5 μm α-naphthalene acetic acid (NAA) after 12 days. A survival rate of 83% was recorded in the greenhouse and the plantlets appeared to be morphologically normal. This is the first report on use of TDZ for in vitro propagation of anchote.
Jane Kahia, Peter Njenga and Margaret Kirika
Jane Kahia, Margaret Kirika, Hudson Lubabali and Sinclair Mantell
Breeding work carried out during the period 1971–85 by the Coffee Research Institute, Ruiru, Kenya resulted in the release of a new improved hybrid Coffea arabica named Ruiru 11. The cultivar combines resistance to coffee berry disease (CBD) and leaf rust, with high yield and good cup quality attributes. The propagation by F1 hybrid seeds production, cuttings, and tip grafting do not produce enough planting materials. There was a need to explore alternative methods and tissue culture offers potential options. The objective of the study was to evaluate the effect of explant sources and cytokinins on induction and regeneration of somatic embryos. Eight different explants were cultured on half-strength Murashige and Skoog (MS) medium supplemented with 10 µm benzylaminopurine (BAP). The effect of kinetin, N6-(2-isopentyl) adenine (2iP) evaluated at (0, 0.5, 5, or 25 µm) or thidiazuron (TDZ) (0, 0.5, 1.0, or 5 µm) added in separate experiments was also evaluated. The percentage of embryogenic cultures and the numbers of embryos per explant were determined after 3 months’ culture. The explant type had a significant effect (P > 0.05) on the induction of somatic embryos. Explants from in vitro-germinated seedlings produced the highest embryogenic cultures (90%) and the highest mean number of embryos (19.36) per explant. Cytokinins strongly enhanced induction and regeneration of somatic embryos. TDZ at 1 µm produced the highest embryogenic cultures (100%) and the highest mean number of embryos (24.2). The embryos were germinated on half-strength MS medium without any hormones. A high (98%) survival rate of the regenerated plantlets was recorded over all the treatments in the greenhouse. This is the first report on induction of high-frequency direct somatic embryos from coffee juvenile tissues. This is of great significance in tissue culture and indeed molecular biology manipulations because it allows regeneration of coffee from several explants.
Jane Kahia, Peter Kanze Sallah, Lucien Diby, Christophe Kouame, Margaret Kirika, Simeon Niyitegeka and Theodore Asiimwe
Cyphomandra betacea (Cav.) is commonly known as Tamarillo or tree tomato. This species is mainly used for its edible fruits which have a high nutritional value and contain relatively high amounts of proteins, vitamins B6, C, E, and provitamin A. The cultivation of Tamarillo in Rwanda is facing major challenges caused mainly by viral diseases such as Tamarillo mosaic virus (TaMV). These diseases are difficult to control and are transferred through vegetative propagation, often resulting in heavy productivity losses and poor-quality fruits. Thus, this study was conducted to evaluate the possibility of using tissue culture as an alternative propagation method. Tamarillo seeds were sterilized using a commercial bleach and germinated in vitro to get clean starting explants. Explants (hypocotyls, leaves, and roots) were cultured on semisolid Murashige and Skoog (MS) media supplemented with 6-benzylaminopurine (BA), N6-2-isopentyl adenine (2iP), 6-furfurylaminopurine (kinetin) evaluated at 5, 10, 20, 40 µM, and thidiazuron (TDZ), evaluated at 0.1, 0.5. 1.0 1.5 µM in separate experiments. Data were collected on the number of microshoots and roots 2 months after culture and analyzed using the Statistical Software for Social Sciences (SPSS) Software version 8. The results showed that the growth regulators evaluated had a significant (P ≤ 0.05) effect on plantlet regeneration from leaf and hypocotyl explants. The media supplemented with BA 40 μM was the most effective in inducing multiple shoots from leaf explants producing 4.67 ± 0.15 shoots per explant. Root explants showed the least morphogenic responses for all the parameters evaluated. The regenerated plantlets were transplanted to the greenhouse and a survival rate of 90% was recorded. During this study, a simple, reproducible, single-step protocol was developed. These results would be useful for mass propagation of Tamarillo.
Modeste Kan Kouassi, Jane Kahia, Christophe N’guessan Kouame, Mathias Gnion Tahi and Edmond Kouablan Koffi
The effect of plant growth regulators on callus and somatic embryogenesis induction in four Cocoa (Theobroma cacao) genotypes was studied. Flower explants were harvested early in the morning and cultured on Driver and Kuniyuki Walnut (DKW) medium supplemented with 1 mg·L−1 of five auxins type (2,4 dichlorophenoxyacetic acid (2,4-D), 3,4 dichlorophenoxyacetic acid (3,4-D), 2,4,5 trichlorophenoxyacetic acid (2,4,5-T), 4-amino-3,5,6-trichloropicolinic acid (picloram), and 3,6-dichloro-2-methoxybenzoic acid (dicamba) in combination with 0.25 or 0.5 mg·L−1 of two cytokinins type (benzylaminopurine (BAP) and 6-furfurylaminopurine [kinetin (Kin)] in a factorial experiment. The plant growth regulators 2,4-D and 2,4,5-T proved to have a broad spectrum action on somatic embryogenesis induction compared with 3,4-D or picloram. There were no significant differences between the two concentrations of cytokinins. However, Kin was found to be more effective in promoting somatic embryogenesis than BAP. Combining 1 mg·L−1 2,4,5-T or 2,4-D with 0.25 mg·L−1 Kin had a broad spectrum action on embryogenesis induction. On the other hand, combining mg·L−1 picloram with 0.5 mg·L−1 Kin or 1 mg·L−1 3,4-D with 0.25 mg·L−1 Kin was only able to induce somatic embryogenesis in a few of the genotypes evaluated. The protocol developed during the current study differs from earleir works as the callus (derived from explants cultured on DKW media) was taken directly to embryo development media as opposed to earlier works in which the callus was taken through a secondary media before being transferred to an embryo development media.
Margaret W. Kirika, Jane W. Kahia, Lucien N. Diby, Eliud M. Njagi, Colombe Dadjo and Christophe Kouame
Erythrina abyssinica (E. abyssinica) is a multipurpose tree and a well-known medicinal plant which is conventionally propagated mainly by seeds. This method may produce a high degree of genetic variability and consequently decrease the medicinal value of the plant. Besides, the seeds have low germination rate and propagation is restricted to rainy season. Hence, there is need to develop a propagation protocol which produces a uniform plants and one which is not restricted to seasons. The objective of this study was to establish an in vitro propagation protocol for the multiplication of E. abyssinica. Seeds were sterilized and germinated in vitro to get sterile starting explants. Sterilization of the seeds was evaluated using different concentrations of a commercial bleach (JIK) ranging from 10%, 20% to 30% for 25 minutes. Kinetin (2.25, 4.50, 6.75, and 9.0 mg/L), and benzylaminopurine (BAP; 2.15, 4.30, 6.46, and 8.61 mg/L) were evaluated in separate experiments for their effect to induce microshoots from nodal explants. Rooting of the microshoot was carried out using half strength Murashige and Skoog (MS) media supplemented with indolebutyric acid (IBA) (0.20, 0.51, and 1.02 mg/L). Statistical analysis software (SAS) package was used to perform analysis of variance on the data to test the significance of the difference between treatments. The result of the sterilization experiment indicated that 10% JIK gave the highest percentage (55%) of clean seeds. Benzylaminopurine evaluated at 8.61 mg/L gave the highest mean number of microshoots (6.80 ± 1.24) after 28 days. On the other hand, IBA evaluated at 0.51mg/L gave the highest mean root length (6.00 ± 01.85 cm). The regenerated plants were acclimatized in the greenhouse and 65% survival rate was recorded after one month. With the increasing worldwide demand for medicinal plants as an alternative to prescription drugs, ex situ, in situ conservation programs and true to type mass propagation of E. abyssinica could benefit from the findings of this study. This is the first report on micropropagation of E. abyssinica.
Jane Kahia, Siaka Kone, Lucien Diby, Georges Ngoran, Colombe Dadjo and Christophe Kouame
Theobroma cacao L. (cacao) is a major tropical crop, grown for its oil-rich seed, which is used in the manufacture of chocolate, its derivatives, and cosmetics. Cacao is cultivated mainly by smallholders and represents a significant export commodity for some developing countries such as Côte d’Ivoire. It is conventionally propagated by seeds, grafting, and cuttings. Somatic embryogenesis offers an alternative method for propagation where large-scale production of planting materials is possible. In the current study, the effect of different concentrations of 2, 4-dichlorophenoxyacetic acid (2, 4-D) and kinetin on induction of somatic embryogenesis and plantlet regeneration in two cocoa clones (coded as C1 and C14) were evaluated. Flowers were collected early in the morning, sterilized, explants excised and cultured on Driver, and Kuniyuki Walnut (DKW) media supplemented with different concentrations of 2, 4-D (9, 10, and 20 µM) and kinetin (0.5, 1, 2.5, 5, 10, and 25 µM) in separate experiments. The frequently used media in somatic embryogenesis of cacao [DKW supplemented with 0.022 µM thidiazuron (TDZ) and 9 µM 2, 4-D] was used as a control. The results of the study showed that explants cultured on media supplemented with 10 µM 2, 4-D and 5 µM kinetin produced the highest (28.0 ± 1.1) mean number of embryos/explant in C1 and this was a 9-fold increase in the number of embryos compared with the control. Explants cultured on media supplemented with 10 µM 2, 4-D and 2.5 µM kinetin produced the highest (7.0 ± 4.0) mean number of embryos/explant in C14 whereas the explants cultured on media supplemented with 20 µM 2, 4-D and 2.5 µM kinetin gave the highest (22.0 ± 1.7) mean number of embryos in clone C1 and C14. The regenerated embryos were germinated and successfully weaned in the green house with a survival rate of 70% being recorded. The paper describes an improved protocol compared with previous work in terms of embryo recovery and survival rate of the elite clones of cocoa through somatic embryogenesis. The results of the current study confirm that somatic embryogenesis of cacao clones is genotype dependent.