Agrobacteriummediated genetic transformation of yam (Dioscorea rotundata): an important tool for functional study of genes and crop improvement

Published online: 15 Sep 2014Although genetic transformation of clonally propagated crops has been widely studied as a tool for crop improvement and as a vital part of the development of functional genomics resources, there has been no report of any existing Agrobacterium-mediated transformation of yam (Dioscorea spp.) with evidence of stable integration of T-DNA. Yam is an important crop in the tropics and subtropics providing food security and income to over 300 million people. However, yam production remains constrained by increasing levels of field and storage pests and diseases. A major constraint to the development of biotechnological approaches for yam improvement has been the lack of an efficient and robust transformation and regeneration system. In this study, we developed an Agrobacterium-mediated transformation of Dioscorea rotundata using axillary buds as explants. Two cultivars of D. rotundata were transformed using Agrobacterium tumefaciens harboring the binary vectors containing selectable marker and reporter genes. After selection with appropriate concentrations of antibiotic, shoots were developed on shoot induction and elongation medium. The elongated antibiotic-resistant shoots were subsequently rooted on medium supplemented with selection agent. Successful transformation was confirmed by polymerase chain reaction, Southern blot analysis, and reporter genes assay. Expression of gusA gene in transgenic plants was also verified by reverse transcription polymerase chain reaction analysis. Transformation efficiency varied from 9.4 to 18.2% depending on the cultivars, selectable marker genes, and the Agrobacterium strain used for transformation. It took 3–4 months from Agro-infection to regeneration of complete transgenic plant. Here we report an efficient, fast and reproducible protocol for Agrobacterium-mediated transformation of D. rotundata using axillary buds as explants, which provides a useful platform for future genetic engineering studies in this economically important crop

Prevalence and Distribution of Cassava Bacterial Blight in the Kenyan Coast

Cassava (Manihot esculenta Crantz) is one of the staple food crops grown in Kenya. Diseases remain one of the major constraints for cassava production. Apart from other major viral diseaes Cassava mosaic and Cassava brown streak, Cassava bacterial blight (CBB) caused by Xanthomonas axonopodis pv manihotis and Xanthomonas axonopodis pv cassavae are a major constraint in cassava production in Kenya. This study was done to identify the prevalence, distribution, and farmers' knowledge of cassava bacterial blight in the coastal region of Kenya. A survey was conducted involving 250 farmers who were randomly selected from two regions of Kilifi and Taita Taveta counties. Among the 250 farmers interviewed, 61.6 % identified cassava bacterial blight symptoms in their farms. The main varieties found growing in the region were Tajirika, Karembo, Kibandameno, and Shibe which were all confirmed by farmers as susceptible to cassava bacterial blight. During the survey, plant samples were randomly collected in the field. Out of the 70 samples collected, 40 of them were confirmed positive with X.pv manihotis and X.pv cassavae which cause cassava bacterial blight. The study concluded that there is a widespread of cassava bacterial blight in Kilifi and Taita taveta counties. Kilifi County had the highest incidence of 22% with Taita Taveta having the lowest incidence of 13%. Kilifi County had a higher severity of 8% as compared to Taita Taveta which had 5% Severity. Therefore there is a need for a proper management program to be deployed in managing the disease to enhance cassava production in the region

Distribution and molecular diversity of whitefly species colonizing cassava in Kenya

Open Access Journal; Published online: 27 Sep 2021The whitefly, Bemisia tabaci (Gennadium, Hemiptera) has been reported to transmit viruses that cause cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) in many parts of sub-Saharan Africa (SSA). Currently, there is limited information on the distribution, species and haplotype composition of the whitefly populations colonizing cassava in Kenya. A study was conducted in the major cassava growing regions of Kenya to address this gap. Analyses of mitochondrial DNA cytochrome oxidase 1 (mtCO1) sequences revealed the presence of four distinct whitefly species: Bemisia tabaci, Bemisia afer, Aleurodicus dispersus and Paraleyrodes bondari in Kenya. The B. tabaci haplotypes were further resolved into SSA1, SSA2 and Indian Ocean (IO) putative species. The SSA1 population had three haplogroups of SSA1-SG1, SSA-SG2 and SSA1-SG3. Application of KASP genotyping grouped the Bemisia tabaci into two haplogroups namely sub-Saharan Africa East and Southern Africa (SSA-ESA) and sub-Saharan Africa East and Central Africa (SSA-ECA). The study presents the first report of P. bondari (Bondar’s nesting whitefly) on cassava in Kenya. Bemisia tabaci was widely distributed in all the major cassava growing regions in Kenya. The increased detection of different whitefly species on cassava and genetically diverse B. tabaci mitotypes indicates a significant influence on the dynamics of cassava virus epidemics in the field. The study highlights the need for continuous monitoring of invasive whitefly species population on cassava for timely application of management practices to reduce the impact of cassava viral diseases and prevent potential yield losses

Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmerpreferred cassava cultivars

CassavagenetictransformationcapacityisstillmostlyrestrictedtoadvancedlaboratoriesintheUSA,EuropeandChina;anditsimplementationandmaintenanceinAfricanlaboratorieshasremainedscarce.TheimpactoftransgenictechnologiesforgeneticimprovementofcassavawilldependlargelyonthetransferofsuchcapabilitiestoresearchersinAfrica,wherecassavahasanimportantsocioeconomicniche.Amajorconstrainttothedevelopmentofgenetictransformationtechnologiesforcassavaimprovementhasbeenthelackofanefficientandrobusttransformationandregenerationsystem.Despitethesuccessachievedingeneticmodificationoffewcassavacultivars,includingthemodelcultivar60444,transgeniccassavaproductionremainsdifficultforfarmer-preferredcultivars.Inthisstudy,aprotocolforcultivar60444developedatETHZurichwassuccessfullyimplementedandoptimizedtoestablishtransformationoffarmer-preferredcassavacultivarspopularineastAfrica.Theconditionsforproductionandproliferationoffriableembryogeniccalli(FEC)andAgrobacterium-mediatedtransformationwereoptimizedforthreeeastAfricanfarmer-preferredcultivars(Ebwanatereka,KibandamenoandSerere).Ourresultsdemonstratedtransformationefficienciesofabout14–22independenttransgeniclinesper100mgofFECforfarmer-preferredcultivarsincomparisonto28linesper100mgofthemodelcultivar60444.Thepresence,integrationandexpressionofthetransgeneswereconfirmedbyPCR,SouthernblotanalysisandhistochemicalGUSassay.ThisstudyreportstheestablishmentofacassavatransformationplatformatInternationalInstituteofTropicalAgriculture(IITA)hostedbyBioscienceseasternandcentralAfrica(BecA)hubinKenyaandprovidesthebasisfortransferringimportanttraitssuchasvirusresistanceandprolongedshelf-lifetofarmer-preferredcultivarsineastAfrica.Weanticipatethatsuchplatformwillalsobeinstrumentaltotransfertechnologiestonationalagriculturalresearchsystems(NARS)insub-SaharanAfrica

Transcriptome Sequencing Reveals a Complete Genome Sequence of Cowpea Aphid-Borne Mosaic Virus from Passion Fruit in Kenya

Analysis of transcriptome sequencing (RNA-Seq) data revealed a complete Cowpea aphid-borne mosaic virus (CABMV) genome from virus-infected passion fruit in Kenya. We compared it with six complete CABMV genomes, one each from Zimbabwe and Uganda and two each from Brazil and India

Virus-Induced Gene Silencing (VIGS) in Cassava Using Geminivirus Agroclones.

peer reviewedVirus-induced gene silencing (VIGS) is an efficient, low-cost, and rapid functional validation tool for candidate genes in planta. The VIGS approach is particularly suitable to perform reverse genetics studies in crop species. Here we present a detailed method to perform VIGS in cassava, from target gene fragment to agroinoculation and VIGS quantitation

Sequencing wild and cultivated cassava and related species reveals extensive interspecific hybridization and genetic diversity.

Cassava (Manihot esculenta) provides calories and nutrition for more than half a billion people. It was domesticated by native Amazonian peoples through cultivation of the wild progenitor M. esculenta ssp. flabellifolia and is now grown in tropical regions worldwide. Here we provide a high-quality genome assembly for cassava with improved contiguity, linkage, and completeness; almost 97% of genes are anchored to chromosomes. We find that paleotetraploidy in cassava is shared with the related rubber tree Hevea, providing a resource for comparative studies. We also sequence a global collection of 58 Manihot accessions, including cultivated and wild cassava accessions and related species such as Ceará or India rubber (M. glaziovii), and genotype 268 African cassava varieties. We find widespread interspecific admixture, and detect the genetic signature of past cassava breeding programs. As a clonally propagated crop, cassava is especially vulnerable to pathogens and abiotic stresses. This genomic resource will inform future genome-enabled breeding efforts to improve this staple crop