Optimisation of in vitro techniques for cassava brown streak virus elimination from infected cassava clones

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV), is an economically important disease of cassava (Manihot esculenta Crantz) in East Africa. The objective of this study was tooptimise in vitro techniques for CBSV elimination from infected Ugandan cassava cultivars. Using semi-solid halfstrength Murashige and Skoog (MS) basal medium, hormone concentration and heat treatment regimes wereoptimised for micropropagation of farmer’s preferred cassava cultivars and CBSV elimination. Single nodes from young cassava stems were cultured for four weeks on ½MS medium supplemented with 6-benzyl amino purine(BAP) and 2,4-dichlorophenoxy acetic acid (2,4-D). The BAP and 2,4-D concentration (mg l-1) combinations used were 0.0 and 0.0, 0.5 and 0.1, 1.0 and 0.2, 1.5 and 0.3, and 2.0 and 0.4, respectively. The optimum medium was used for in vitro thermotherapy using four temperature regimes, namely 30-34, 34-38, 36-40 and 38-42 oC for 8 hoursdarkness and 16 hours light, respectively, for four weeks. The best plantlet growth in terms of height was observed on MS medium supplemented with 0.5 mg l-1 BAP and 0.1 mg l-1 2,4-D. Highest CBSV elimination efficiency of 40%, with 49% plantlet survival was observed at 36 oC for 8 hours darkness and 40 oC for 16 hours light. These results indicate that in vitro techniques can greatly enhance CBSV elimination and, thus, provide a means of CBSD management through dissemination and conservation of popular but CBSD susceptible cultivars

An International Service Learning Guide for Occupational Therapy Students and Clinicians

We ourselves feel that what we are doing is just a drop in the ocean. But the ocean would be less because of that missing drop -Mother Teresa Increased diversity within the U.S. demands the education of occupational therapy clinicians who can deliver culturally responsive care. The number of immigrants to the U.S. is at an all-time high and the African-American and Hispanic populations are expected to double by mid-century (U.S. Census Bureau, 2008). Occupational therapy students and entry-level clinicians have identified the need for more information, resources, and experience with people from different cultures (Murden et aI., 2008). Service learning and cultural immersion were identified as educational methods that may facilitate the development of cultural competence and lead to culturally sensitive care (Bonder, Martin & Miracle, 2002a; Iwama, 2009; O\u27Grady, 2000; Price et aI., 2005). This scholarly project culminated in a guide, which is intended to provide occupational therapy students and clinicians with the framework necessary to develop and complete an international service learning experience for the promotion of cultural competence. Guided by the Canadian Model of Occupational Performance and Engagement (CMOP-E), the model of service learning, and adult learning theory, this guide addresses the emotional, intellectual, financial, physical, and social elements of service learning to achieve both learner and client goals. The guide is intended for use in planning an international service learning project in a developing country, but may be adapted for use in local or regional areas of the U.S

Occurrence of rice yellow mottle virus resistance breaking isolates in lowland catchment zones of Uganda

Rice (Oryza spp; 2n=24.) production in Uganda and Africa in general, is seriously threatened by the Rice yellow mottle virus disease (RYMVD), a disease caused by Rice yellow mottle virus (RYMV) within the genus Sobemovirus; family Sobemoviridae. This study investigated the existence and distribution of resistance-breaking RYMV pathotype in the three major lowland rice catchment areas in Uganda. Four known rice accessions resistant to Rice yellow mottle virus (RYMV) namely; Gigante, Tog5672, Tog5674 and Tog5681, carrying resistant allele’s rymv1-2, rymv1-4 & RYMV3, rymv1-5 and rymv1-3, respectively, were tested for their response to different RYMV isolates. The isolates were collected from three major lowland rice catchment areas of Doho, Kibimba, and Olweny in Uganda. Out of 100 leaf samples collected from the field and assayed for RYMV and confirmed to be positive using RT-PCR, 83 isolates induced symptoms on IR64- the RYMV susceptible line. Seventy-seven (92.8%) isolates were able to overcome resistance in at least one of the four differential rice accessions, as confirmed by the presence of RYMV symptoms; while 6 (7.2%) isolates were asymptomatic. Variation in time (days) for symptom development post-inoculation (dpi) and AUDPC were observed. Symptoms appeared within 5-7 days on IR64; while it took on average 11, 18, 36, and 18 days to appear on Gigante, Tog5672, Tog5674 and Tog5681, respectively. The highest AUDPC was observed on IR64 (254.7); while the lowest was observed on Tog5681 (74.1). Two major patho-groups were observed; those that broke down resistance in Gigante only (25.3%) and Gigante & Tog5672 (33.7%). Five isolates from Doho (Budaka & Bugiri districts) and Kibimba (Butaleja district) catchment areas broke down RYMV resistance in three accessions i.e. (Tog5681, Gigante & Tog5672) and (Tog5674, Gigante & Tog5672), respectively. Resistance breaking isolates were confirmed in all  the three sampled catchment zones, however, Doho and Kibimba had some unique isolates that broke down resistance in accessions carrying resistance allele rymv 1-3 and rymv1-5 in addition to rymv1-2. Results from this study showed that RYMV isolates in Uganda can break down resistance conferred by the rymv1-2 resistance gene allele. However, accessions Tog5681 and Tog5674 seem to hold stable RYMV resistance and, thus are recommended for RYMV breeding

Production of friable embryogenic callus and regeneration of Ugandan farmer-preferred cassava genotypes

Generation of embryogenic callus is a key step in genetic engineering of many crop species, including cassava. Protocols for generation of friable embryogenic callus (FEC) have been lacking for Ugandan cassava genotypes, thereby delaying their genetic engineering for agronomic and other desirable traits. The objective of this study was to determine conditions suitable for production and regeneration of FEC in the Ugandan cassava genotypes; Aladu, Bukalasa and Ebwanateraka, and control cultivar 60444. Immature leaf lobe explants were established on Murashige and Skoog (MS) based media for initiation of organized embryogenic callus (OES). To produce FEC, resulting OES were established on Gresshoff and Doy based callus induction media with varying levels of sucrose, maltose, tyrosine, tryptophan, naphthalene acetic acid (NAA) under light and dark conditions. Subsequently, FEC was subcultured to MS-based embryo maturation and embryo regeneration media. All genotypes produced OES. All genotypes produced FEC except Bukalasa. The amino acid tyrosine favoured production of FEC in Aladu and Ebwanatereka, but not in 60444, while 20 g/L of sucrose trigged production of FEC in Aladu and 60444, but 40 g/L of sucrose was superior for Ebwanatereka. Media supplemented with 1 ml/L naphthalene acetic acid NAA facilitated embryo regeneration in Ebwanatereka and 60444, while Aladu responded better to 5 ml/L NAA. Light, tyrosine and sucrose were essential for FEC production in Uganda cultivars while NAA was required for regeneration of somatic embryos. Ability to produce FEC in these genotypes lays a foundation for their improvement through genetic transformation for the desired and agronomic traits.Key words: Cassava (Manihot esculenta Crantz), somatic embryogenesis, amino acids, carbon sources

Optimisation of in vitro techniques for Cassava brown streak virus elimination from infected cassava clones

Cassava brown streak disease (CBSD), caused by Cassava brown streak virus (CBSV), is an economically important disease of cassava ( Manihot esculenta Crantz) in East Africa. The objective of this study was to optimise in vitro techniques for CBSV elimination from infected Ugandan cassava cultivars. Using semi-solid half-strength Murashige and Skoog (MS) basal medium, hormone concentration and heat treatment regimes were optimised for micropropagation of farmer's preferred cassava cultivars and CBSV elimination. Single nodes from young cassava stems were cultured for four weeks on 1/2MS medium supplemented with 6-benzyl amino purine (BAP) and 2,4-dichlorophenoxy acetic acid (2,4-D). The BAP and 2,4-D concentration (mg 1-1) combinations used were 0.0 and 0.0, 0.5 and 0.1, 1.0 and 0.2, 1.5 and 0.3, and 2.0 and 0.4, respectively. The optimum medium was used for in vitro thermotherapy using four temperature regimes, namely 30-34, 34-38, 36-40 and 38-42 \ub0C for 8 hours darkness and 16 hours light, respectively, for four weeks. The best plantlet growth in terms of height was observed on MS medium supplemented with 0.5 mg 1-1 BAP and 0.1 mg 1-1 2,4-D. Highest CBSV elimination efficiency of 40%, with 49% plantlet survival was observed at 36 \ub1C for 8 hours darkness and 40 oC for 16 hours light. These results indicate that in vitro techniques can greatly enhance CBSV elimination and, thus, provide a means of CBSD management through dissemination and conservation of popular but CBSD susceptible cultivars.La maladie de la striure brune du manioc (CBSD) caus\ue9e par le virus de la striure brune (CBSV), est une maladie \ue9conomiquement importante du manioc ( Manihot esculenta Crantz) en Afrique de l'Est. L'objectif de cette \ue9tude \ue9tait d'optimiser les techniques in vitro pour l'\ue9limination du CBSV des cultivars ougandais infect\ue9s. En utilisant le m\ue9dium basal semi-solid half strength Murashige and Skoog, la concentration d'hormone et les regimes de traitement de chaleur \ue9taient optimis\ue9s pour la micropropagation des cultivars de manioc pr\ue9f\ue9r\ue9s par les fermiers et l'\ue9limination du CBSV. Les nodes simples des jeunes tiges de manioc \ue9taient cultiv\ue9s pendant quatre semaines sur le m\ue9dium 1/2 MS suppl\ue9ment\ue9s avec 6-benzyl amino purine (BAP) et 2,4-dichlorophenoxy acetic acid. Les combinaisons de concentrations (mg 1-1) de BAP et de 2.4-D utilis\ue9es \ue9taient 0.0 et 0.0, 0.5 et 0.1, 1.0 et 0.2, 1.5 et 0.3, et 2.0 et 0.4, respectivement. Le m\ue9dium optimum \ue9tait utilis\ue9 pour la thermoth\ue9rapie utilisant quatre regimes de temp\ue9rature dont 30-34, 34-38, 36-40 et 38-42 \ub0C pendant 8 heures en obscurit\ue9 et 16 heures sous lumi\ue8re, respectivement pendant quatre semaines. La meilleure croissance en hauteur des plantules \ue9tait observ\ue9e sur le m\ue9dium MS suppl\ue9ment\ue9 avec 0.5 mg 1-1 BAP et 0.1 mg 1-1 2,4-D. L'efficience la plus \ue9lev\ue9e de 40 % en terme d'\ue9limination du CBSV, avec 49 % de la survie de plantules \ue9tait observ\ue9e \ue0 36 \ub0C pendant 8 heures sous obscurit\ue9 et 40 \ub0C pendant 16 heures sous lumi\ue8re. Les r\ue9sultats indiquent que les techniques in vitro peuvent grandement promouvoir l'\ue9limination du CBSV et offrent ainsi un moyen de gestion de CBSD \ue0 travers la dissemination et la conservation des cultivars aussi populaires que susceptibles au SBSD

Efficient CRISPR/Cas9 genome editing of Phytoene desaturase in cassava

CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analyzed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90 to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence analysis revealed that 38/38 (100%) of the plant lines examined carried mutations at the targeted MePDS site, with insertions, deletions, and substitutions recorded. One putatively mono-allelic homozygous line (1/19) was found from cv. 60444, while 1 (1/19) and 4 (4/19) putatively bi-allelic homozygous lines were found in 60444 and TME204, respectively. The remaining plant lines, comprised mostly of the chimeras, were found to be putatively heterozygous. We observed minor (1 bp) nucleotide substitutions and or deletions upstream of the 5 0 and or downstream of the 3 0 targeted MePDS region. The data reported demonstrates that CRISPR/Cas9-mediated genome editing of cassava is highly efficient and relatively simple, generating multi-allelic mutations in both cultivars studied. Modification of MePDS described here generates visually detectable mutated events in a relatively short time frame of 6-8 weeks, and does not require sequencing to confirm editing at the target. It therefore provides a valuable platform to facilitate rapid assessment and optimization of CRISPR/Cas9 and other genome-editing technologies in cassava

Occurrence of rice yellow mottle virus resistance breaking isolates in lowland catchment zones of Uganda

Rice ( Oryza spp; 2n=24.) production in Uganda and Africa in general, is seriously threatened by the Rice yellow mottle virus disease (RYMVD), a disease caused by Rice yellow mottle virus (RYMV) within the genus Sobemovirus; family Sobemoviridae. This study investigated the existence and distribution of resistance-breaking RYMV pathotype in the three major lowland rice catchment areas in Uganda. Four known rice accessions resistant to Rice yellow mottle virus (RYMV) namely; Gigante, Tog5672, Tog5674 and Tog5681, carrying resistant allele\u2019s rymv1-2, rymv1-4 & RYMV3, rymv1-5 and rymv1-3, respectively, were tested for their response to different RYMV isolates. The isolates were collected from three major lowland rice catchment areas of Doho, Kibimba, and Olweny in Uganda. Out of 100 leaf samples collected from the field and assayed for RYMV and confirmed to be positive using RT-PCR, 83 isolates induced symptoms on IR64- the RYMV susceptible line. Seventy-seven (92.8%) isolates were able to overcome resistance in at least one of the four differential rice accessions, as confirmed by the presence of RYMV symptoms; while 6 (7.2%) isolates were asymptomatic. Variation in time (days) for symptom development post-inoculation (dpi) and AUDPC were observed. Symptoms appeared within 5-7 days on IR64; while it took on average 11, 18, 36, and 18 days to appear on Gigante, Tog5672, Tog5674 and Tog5681, respectively. The highest AUDPC was observed on IR64 (254.7); while the lowest was observed on Tog5681 (74.1). Two major patho-groups were observed; those that broke down resistance in Gigante only (25.3%) and Gigante & Tog5672 (33.7%). Five isolates from Doho (Budaka & Bugiri districts) and Kibimba (Butaleja district) catchment areas broke down RYMV resistance in three accessions i.e. (Tog5681, Gigante & Tog5672) and (Tog5674, Gigante & Tog5672), respectively. Resistance breaking isolates were confirmed in all \ua0the three sampled catchment zones, however, Doho and Kibimba had some unique isolates that broke down resistance in accessions carrying resistance allele rymv 1-3 and rymv1-5 in addition to rymv1-2. Results from this study showed that RYMV isolates in Uganda can break down resistance conferred by the\ua0rymv1-2 resistance gene allele. However, accessions Tog5681 and Tog5674 seem to hold stable RYMV resistance and, thus are recommended for RYMV breeding.La production de riz ( Oryza spp ; 2n=24.) en Ouganda et en Afrique en g\ue9n\ue9ral, est s\ue9rieusement menac\ue9e par la maladie du virus de la marbrure jaune du riz (RYMVD), une maladie caus\ue9e par le virus de la marbrure jaune du riz (RYMV) du genre Sobemovirus ; famille des Sobemoviridae. Cette \ue9tude a examin\ue9 l\u2019existence et la distribution du pathotype RYMV qui brise la r\ue9sistance dans les trois principaux bassins versants de riz de plaine en Ouganda. Les quatre accessions de riz connues r\ue9sistantes au \ua0virus\ua0de la panachure\ua0jaune du riz\ua0(RYMV, Rice yellow mottle\ua0virus) \ue0 savoir; Gigante, Tog5672, Tog5674 et Tog5681, portant respectivement les all\ue8les r\ue9sistants rymv1-2, rymv1-4 & rymv3, rymv1-5 et rymv1-3, ont \ue9t\ue9 test\ue9s pour leur r\ue9ponse \ue0 diff\ue9rents isolats de RYMV. Les isolats ont \ue9t\ue9 collect\ue9s dans les trois principaux bassins versants du riz de plaine de Doho, Kibimba et Olweny en Ouganda. Sur les 100 \ue9chantillons des feuilles pr\ue9lev\ue9s sur le terrain et test\ue9s pour le RYMV et confirm\ue9s positifs par RT-PCR, 83 isolats ont induit des sympt\uf4mes sur IR64, la lign\ue9e sensible au RYMV. Les soixante-dix-sept (92,8 %) isolats ont r\ue9ussi \ue0 surmonter la r\ue9sistance dans au moins une des quatre accessions du riz diff\ue9rentielles, comme le confirme la pr\ue9sence des sympt\uf4mes du RYMV\ua0; tandis que 6 (7,2 %) isolats \ue9taient asymptomatiques. Une variation dans le temps (jours) pour le d\ue9veloppement des sympt\uf4mes apr\ue8s l\u2019inoculation (dpi, development post-inoculation) et l\u2019AUDPC a \ue9t\ue9 observ\ue9e. Les sympt\uf4mes sont apparus dans les 5 \ue0 7 jours sur IR64\ua0; alors qu\u2019il a fallu en moyenne 11, 18, 36 et 18 jours pour appara\ueetre sur Gigante, Tog5672, Tog5674 et Tog5681, respectivement. L\u2019AUDPC le plus \ue9lev\ue9 a \ue9t\ue9 observ\ue9 sur IR64 (254,7); tandis que l\u2019AUDPC le plus bas a \ue9t\ue9 observ\ue9 sur Tog5681 (74,1). Deux patho-groupes principaux ont \ue9t\ue9 observ\ue9s; ceux qui ont bris\ue9 la r\ue9sistance dans les Gigante uniquement (25,3%) et Gigante & Tog5672 (33,7%). Les cinq isolats des bassins versants de Doho (districts de Budaka et Bugiri) et de Kibimba (district de Butaleja) ont bris\ue9 la r\ue9sistance au RYMV dans trois accessions, \ue0 savoir (Tog5681, Gigante et Tog5672) et (Tog5674, Gigante et Tog5672), respectivement. Des isolats brisant la r\ue9sistance ont \ue9t\ue9 confirm\ue9s dans les trois zones de captage \ue9chantillonn\ue9es, cependant, Doho et Kibimba avaient des isolats uniques qui brisaient la r\ue9sistance dans des accessions portant les all\ue8les de r\ue9sistance rymv1-3 et rymv1-5 en plus de rymv1-2. Les r\ue9sultats de cette \ue9tude ont montr\ue9 que les isolats de RYMV en Ouganda peuvent briser la r\ue9sistance conf\ue9r\ue9e par l\u2019all\ue8le du g\ue8ne de r\ue9sistance rymv1-2. Cependant, les accessions Tog5681 et Tog5674 semblent d\ue9tenir une r\ue9sistance stable au RYMV et sont donc recommand\ue9es pour la s\ue9lection du RYMV