Rate of re-infection of tissue culture-derived Latin American and East and Southern African cassava genotypes by mosaic disease

The rate of reinfection by cassava mosaic disease (CMD) in initially virus-free cassava plants of two Latin American and twelve East and Southern African cassava genotypes grown was studied under high disease pressure conditions. An improved clone, TMS 4(2)1425, from the International Institute of Tropical Agriculture was used as check. The virus-free plants had been produced through meristem-tip culture and multiplied in a pest-proof screen house. The genotypes were planted in single row plots of 5 plants each, arranged in a randomized complete block design with 4 replications and spacing of 1 × 1 m2. Incidence and severity of CMD on the genotypes were assessed weekly, from 4 to 16 weeks after planting (WAP). Enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) tests for the cassava mosaic virus were carried out using young leaves collected randomly at 15, 16 and 17 WAP from plants both with and without symptoms. Six genotypes had > 60% CMD incidence at 4 WAP; by 7 WAP, 12 genotypes had > 60% incidence. Only Kigoma red, Kiroba, and UKG-41-6 were notinfected at 4 WAP while Mbudumali had 90% incidence at this time. At 16 WAP, ten genotypes had 100% CMD incidence; Kigoma Red was 39.6% infected. ELISA detected a mean CMD reinfection rate of 66.6%; PCR detected 69%. A high negative and significant (P< 0.01) correlation (r = - 0.70) was established between CMD severity and storage root yield.Keywords: Virus-free cassava genotypes, tissue culture, rate of reinfection, cassava mosaic diseas

Induction of somatic embryogenesis in recalcitrant sweetpotato (Ipomoea batatas L.) cultivars

Genetic transformation is considered as one of the most promising options for improvement of crop traits. Current transformation methods for sweetpotato depend on plant regeneration through organogenesis or somatic embryogenesis. Somatic embryogenesis and plant regeneration at a high frequency has been  restricted to a few sweetpotato varieties. Three auxins namely: 2,4-dichlorophenoxyacetic acid (2,4-D),  4-fluoroamphetamine (4-FA) and 4,5-trichlorophenoxyacetic acid (2,4,5-T) were investigated in this study for enhancing somatic embryogenesis from various plant organs of recalcitrant African sweetpotato cultivars.  2,4-D was found to be the best (p . 0.05) for induction of embryogenic callus. Cultivar Bwanjule had the highest  (20.2%) embryogenic callus frequency among the five African cultivars tested. The highest number of plants in this study was regenerated from the non-African cultivar variety Jonathan on media supplemented with 0.2 mg Zeatin. The emergence of roots from callus of recalcitrant Ugandan cultivars and the comparable high embryogenic responses in this work demonstrate the potential for regenerating plants from African  cultivars that have not been regenerated before. The regeneration of roots in this work could be useful for the initiation of root cultures. The most important application of this work is in genetic transformation of sweet potato, particularly for improvement of resistance to weevils.Key words: Embryogenesis, plant growth regulators, plant regeneration, Ipomoea batatas

THIDIAZURON IMPROVES ADVENTITIOUS BUD AND SHOOT REGENERATION IN RECALCITRANT SWEETPOTATO

Induction of adventitious buds and shoots from intact leaves and stem internode segments of two recalcitrant Ugandan sweetpotato ( Ipomoea batatas L.) cultivars was investigated in vitro on Murashige and Skoog (MS) medium, supplemented with 3 different levels (0.5, 2.0 and 4.0 \u3bcM) of Thidiazuron (TDZ). Shoots were regenerated in all TDZ concentrations in cvs. Kyebandula and Bwanjule. The inclusion of 0.25 \u3bcM \u3b1-Naphthalene acetic acid (NAA) in MS medium, containing TDZ (0.5 \u3bcM), improved shoot regeneration frequency from 12.1 to 22.6% for cv. Kyebandula stems and from 21.61 to 42.9% for cv. Bwanjule stems. However, there was about 10% reduction in adventitious bud induction frequency for both cultivars, when NAA was included in the medium. The highest frequency (66.7%) of adventitious bud induction was achieved from stem explants of cv. Kyebandula. The conversion of adventitious buds into shoots was improved when TDZ was reduced or completely removed in subsequent stages of culture. The number of explants forming shoots was significantly (P<0.001) higher when stem explants were cultured for 7 days on TDZ-supplemented MS medium before transfer to TDZfree MS medium supplemented with NAA. Stem internode pieces from position 3 were the best (70.0%) in adventitious bud formation. However, most buds (76.2%) were not converted to shoots. The most important application of the de novo regeneration protocol developed in this study is in genetic transformation for improvement of sweetpotato productivity.L\u2019 induction des bourgeons adventices et des pousses \ue0 partir des feuilles intacts et des tiges des segments internodes de deux patates douces ( Ipomoea batatas L.) Ugandaises recalcitrantes \ue9tait \ue9tudi\ue9e en milieu in vitro sur le m\ue9dia Murashige et Skoog (MS) suppl\ue9ment\ue9 avec 3 niveaux diff\ue9rents (0.5, 2.0 et 4.0 \u3bcM) de Thidiazuron (TDZ). Les pousses \ue9taient r\ue9g\ue9n\ue9r\ue9es dans toutes les concentrations TDZ dans les cvs. Kyebandula et Bwanjule. L\u2019inclusion de 0.25 \u3bcM \u3b1-Naphthal\ue8ne d\u2019 acide ac\ue9tique (NAA) dans le m\ue9dia MS contenant le TDZ (0.5 \u3bcM) a am\ue9lior\ue9 la fr\ue9quence de la r\ue9n\ue9g\ue9n\ue9ration des pousses de 12.1 \ue0 22.6% pour les pousses cv. Kyebandula et de 21.61 \ue0 42.9% pur les pousses cv. Bwanjule. Cependant, il y a eu environ 10% de r\ue9duction en termes de la fr\ue9quence d\u2019induction des bourgeons adventices pour les deux cultivars lorsque NAA \ue9tait inclu dans le m\ue9dia. La fr\ue9quence la plus \ue9lev\ue9e (66.7%) d\u2019induction des bourgeons adventices \ue9tait r\ue9alis\ue9e des explants des tiges de cv. Kyebandula. La conversion des bourgeons adventices en pousses \ue9tait am\ue9lior\ue9e lorsque TDZ \ue9tait r\ue9duit ou compl\ue8tement enlev\ue9 des \ue9tapes de cultures subs\ue9quentes. Le nombre d\u2019explants formant les pousses \ue9tait significativement (P<0.001) sup\ue9rieur lorsque les explants des tiges \ue9taient cultiv\ue9s pendant 7 jours sur le m\ue9dia MS suppl\ue9ment\ue9 par TDZ avant le transfert dans le m\ue9dia MS sans TDZ avec pour suppl\ue9ment NAA. Les morceaux de tiges internodes de la position 3 \ue9taient le meilleur (70.0%) en formation de bourgeons adventices. Cependant, plus de bourgeons (76.2%) n\u2019\ue9taient pas converties en pousses. L\u2019application la plus importante du protocol de la r\ue9g\ue9n\ue9ration de novo d\ue9v\ue9lopp\ue9e dans cette \ue9tude reside dans la transformation g\ue9n\ue9tique pour l\u2019am\ue9lioration de la productivit\ue9 de la patate douce

TRANSIENT EXPRESSION OF \u3b2-GLUCURONIDASE IN RECALCITRANT UGANDAN SWEETPOTATO AND PUTATIVE TRANSFORMATION WITH TWO CRY GENES

Sweetpotato ( Ipomoea batatas Lam.) has high potential to contain hunger, malnutrition and poverty in sub-Saharan Africa (SSA), since it gives early yield with few inputs. However, productivity of the crop in Africa is very low due to various challenges, such as severe viral diseases and increasing attacks by sweetpotato weevils, Cylas puncticollis and C. brunneus . Effective resistance to weevils has not been identified in the sweetpotato gene pool. On the other hand, the weevil-resistance genes, cry7Aa1 and cry3Ca1 were assembled into a plasmid vector for use in genetic transformation of African sweetpotato cultivars. The parameters for efficient transfer of these genes and the conditions for de novo regeneration optimised in preliminary studies were used in the genetic transformation of Ugandan landrace \u2018Kyebandula\u2019 with Agrobacterium tumefaciens EHA 105 harbouring the plasmid pCIP84, which contains cry7Aa1, cry3Ca1 and nptII in its T-DNA. Fifty-four percent of the explants formed adventitious buds. With a mean of 7 buds formed per explant, 6.0% explants formed shoots with a mean of one shoot per explant for those explants that formed shoots on medium containing 50 mg L-1 kanamycin as a selection agent. PCR analysis using primers for cry7Aa1 showed that the transformation efficiency could be as high as 2%. These data highlight the potential of genetic transformation in transferring resistance genes and pave way for enhancement of food security through production of adapted sweetpotato weevil resistant cultivars.La patate douce ( Ipomoea batatas Lam.) a un potentiel de contenir la faim, la malnutrition et la pauvret\ue9 en Afrique Sub Saharienne \ue9tant donn\ue9 sa pr\ue9cocit\ue9 et son grand rendement avec peu d\u2019intrants. Par ailleurs, sa productivit\ue9 est trop basse due aux diverses contraintes li\ue9es aux maladies virales et attaques sans cesse croissante des charan\ue7ons Cylas puncticollis and C. brunneus . Une r\ue9sistance efficace au charan\ue7on n\u2019a pas encore \ue9t\ue9 identifi\ue9e dans la collection des g\ue8nes de la patate douce. D\u2019autre part, les g\ue8nes de r\ue9sistance aux charan\ue7ons, cry7Aa1 et cry3Ca1 \ue9taient assembl\ue9es un vecteur de plasmide pour utilisation dans la transformation g\ue9n\ue9tique des cultivars de patate douces africaines. Les param\ue8tres pour transfert d\u2019efficacit\ue9 de ces g\ue8nes et les conditions de r\ue9g\ue9n\ue9ration de novo optimis\ue9es dans des \ue9tudes pr\ue9liminaires \ue9taient utilis\ue9es dans la transformation g\ue9n\ue9tique du landrace ougandais \u2018Kyebandula\u2019avec l\u2019 Agrobacterium tumefaciens EHA 105 portant le plasmide pCIP84, contenant les cry7Aa1, cry3Ca1 et nptII dans son T-ADN. Cinquante quatre pourcent des explants ont form\ue9 des bourgeons adventices. Avec une moyenne de 7 bourgeons par explant, 6.0% d\u2019explants ont form\ue9 des tiges avec une moyenne d\u2019une tige par explant pour ces explants qui ont form\ue9 des tiges sur le media contenant 50 mg L-1 de kanamycine comme agent de s\ue9lection. L\u2019analyse par PCR utilisant des primers pour cry7Aa1 a montr\ue9 que la transformation efficace pourrait \ueatre aussi \ue9lev\ue9e que 2%. Ces donn\ue9es soulignent le potentiel de transformation g\ue9n\ue9tique dans le transfert des g\ue8nes de r\ue9sistance et un moyen d\u2019am\ue9liorer la s\ue9curit\ue9 alimentaire \ue0 travers la production des cultivars de r\ue9sistance adapt\ue9e aux charan\ue7ons de la patate douce

EVALUATION OF BIOSSAYS FOR TESTING Bt SWEETPOTATO EVENTS AGAINST SWEETPOTATO WEEVILS

Sweetpotato weevil ( Cylas puncticollis ) Boheman is a serious pest throughout Sub-Saharan Africa region and is a big threat to sweetpotato cultivation. Ten transgenic sweetpotato events expressing Cry7Aa1, Cry3Ca1, and ET33-34 proteins from Bacillus thuringiensis (Bt) were evaluated for resistance against C. puncticollis. Four bioassays used were: (i) 1st instar larva in an artificial diet using root powder of transgenic events, (ii) whole root of transgenic events infested with female adults, (iii) root chip, and (iv) small root of transgenic events both infested with egg-plugs. DAS-ELISA analysis showed variation in Cry protein concentration among the events that ranged between 0.1 and 0.394 \ub5g g-1 of root fresh weight. The highest protein quantity was observed in the event carrying the ET33-34 transcript. In general, transgenic events had no significant effect on larval survival (P = 0.28) and pupal (P = 0.86) development, though maximum pupation of 96% was observed on event CIP410009.12 that had very low expression levels of cry3Ca1 gene. Root chips were prone to damage by fungi and desiccation especially during the first larval instar. The whole root bioassay had less handling injuries to weevils compared with other methods. However, it requires a large number of adult females for oviposition and roots per event to be tested to obtain efficacy results with statistical rigour. The small root egg plug bioassay required much fewer roots and experimental insects to assess larval mortality and development. The root chip method was the least desirable because of susceptibility to fungal and bacterial contamination. In addition, this method was the most labour intensive in terms of frequent replacement of root chips for weevil development. Hence, the most appropriate method for testing Bt efficacy in sweetpotato is the small root egg-plug bioassay. Nonetheless, none of the transgenic events tested provided weevil control probably because of low Cry protein expression in storage roots.La charan\ue7on de la patate douce ( Cylas puncticollis ) Boheman constitue une importante peste \ue0 travers la r\ue9gion d\u2019Afrique sub-saharienne et une grande contrainte \ue0 la production de la culture. Dix patate douce transg\ue9niques d\u2019\ue9v\ue9n\ue9ments exprimant les prot\ue9ines Cry7Aa1, Cry3Ca1, et ET33-34 des Bacillus thuringiensis (Bt) \ue9taient \ue9valu\ue9es pour r\ue9sistance contre C. puncticollis. Quatre bioassais utilis\ue9s \ue9taient: (i) une larve de premier instar dans une alimentation artificielle utilisant une poudre racinaire d\u2019\ue9v\ue9nement transg\ue9nique, (ii) racine entire d\u2019\ue9v\ue9nement transg\ue9niques infest\ue9s avec adultes femelles, (iii) un morceau racinaire, et (iv) petite racine d\u2019\ue9v\ue9nements transg\ue9niques tous infest\ue9s avec des oeufs. L\u2019analyse DAS-ELISA a montr\ue9 une variation dans la concentration en protein Cry parmi les \ue9v\ue9nements variant de 0.1 \ue0 0.394 \ub5g g-1 de poids de raciness fra\ueeches. La quqntit\ue9 la plus \ue9lev\ue9e de prot\ue9ines \ue9tait observ\ue9e dans l\u2019\ue9v\ue9nement portant le relev\ue9 ET33-34. En g\ue9n\ue9ral, les \ue9v\ue9nements transg\ue9niques n\u2019avaient aucun effet significatif sur la survie de larves (P = 0.28) et le d\ue9veloppement de la nimphe (P = 0.86), bien que la pupation maximale de 96% \ue9tait observ\ue9e sur l\u2019\ue9v\ue9nement CIP410009.12 qui avait un tr\ue8s bas niveau d\u2019expression du g\ue8ne cry3Ca1. Les morceaux de raciness \ue9taient susceptibles d\u2019\ueatre endommag\ue9s par les champignons et la dessication sp\ue9cialement durant le premier instar larvaire. La racine enti\ue8re bioassay \ue9tait moins affct\ue9e par la charan\ue7on et avait moins de blessures en comparaison \ue0 d\u2019autres m\ue9thodes. Par ailleurs, il n\ue9cessite un grand nombre d\u2019adultes femelles pour l\u2019oviposition et raciness par \ue9v\ue9nement devrant \ueatre test\ue9s pour obtenir des r\ue9sultats efficaces avec rigueur statistique. Le bioassai de petits oeufs racinaires ongt n\ue9cessit\ue9 un peu moins de raciness et insectes exp\ue9rimentaux pour \ue9valuer la mortalit\ue9 larvaire et leur d\ue9veloppement. La m\ue9thode de morceaux racinaires \ue9tait la moins desirable \ue0 cause de la contamination fongique et bact\ue9rienne. En plus, cette m\ue9thode exigeait plus de travail intensif en terme remplacement fr\ue9quent de morceaux de racines pour le d\ue9veloppement de charan\ue7on. Ainsi, la m\ue9thode la plus appropri\ue9e pour tester l\u2019efficacit\ue9 du Bt dans la patate douce est le bioassai de petis oeufs racinaires. N\ue9amoins, aucun des \ue9v\ue9nements transg\ue9niques test\ue9s n\u2019a fourni un control probable de la charan\ue7on suite \ue0 une basse expression de la protein Cry dans les raciness de stockage

Identification of simple sequence repeat markers for sweetpotato weevil resistance

The development of sweetpotato [Ipomoea batatas (L.) Lam] germplasm with resistance to sweetpotato weevil (SPW) requires an understanding of the biochemical and genetic mechanisms of resistance to optimize crop resistance. The African sweetpotato landrace, ‘New Kawogo’, was reported to be moderately resistant to two species of SPW, Cylas puncticollis and Cylas brunneus. Resistance has been associated with the presence of hydroxycinnamic acids esters (HCAs), but the underlying genetic basis remains unknown. To determine the genetic basis of this resistance, a bi-parental sweetpotato population from a cross between the moderately resistant, white-fleshed ‘New Kawogo’ and the highly susceptible, orange-fleshed North American variety ‘Beauregard’ was evaluated for SPW resistance and genotyped with simple sequence repeat (SSR) markers to identify weevil resistance loci. SPW resistance was measured on the basis of field storage root SPW damage severity and total HCA ester concentrations. Moderate broad sense heritability (H2 = 0.49) was observed for weevil resistance in the population. Mean genotype SPW severity scores ranged from 1.0 to 9.0 and 25 progeny exhibited transgressive segregation for SPW resistance. Mean genotype total HCA ester concentrations were significantly different (P < 0.0001). A weak but significant correlation (r = 0.103, P = 0.015) was observed between total HCA ester concentration and SPW severity. A total of five and seven SSR markers were associated with field SPW severity and total HCA ester concentration, respectively. Markers IBS11, IbE5 and IbJ544b showed significant association with both field and HCA-based resistance, representing potential markers for the development of SPW resistant sweetpotato cultivars

Evaluation and delivery of disease-resistant and micronutrientdense sweetpotato varieties to farmers in Uganda

Uganda is among the African countries reported to be at high risk of Vitamin A deficiency (VAD) with prevalence of VAD among children and women at 28 and 23%, respectively. Promoting orange-fleshed sweetpotato (OFSP) (Ipomoea batatas) rich in Vitamin A will play a significant role in combating VAD. However, sweetpotato virus disease is a major constraint to OFSP. The National Crops Resources Research Institute (NaCRRI) under took research on improvement, evaluation and delivery of promising OFSP to farmers. Selected parents with complimentary traits are planted to a crossing block annually and true seed is harvested and used to establish nurseries the plants of which are individually screened for reaction to SPVD, Alternaria blight, and depth of the orange colour in the root flesh. Promising selections are evaluated through a series of verification multi-locational trials on-station. Selections are subsequently advanced onfarm to test their performance under farmer managed conditions and for their acceptability by farmers and consumers for both food and other uses. To date nine OFSP varieties have been released and disseminated to farmers.Generally, most of the released OFSP varieties are susceptible to weevils (Cylas spp.), but moderately resistant to both SPVD and Alternaria blight