Somatic embryogenesis in sweetpotato is highly genotype dependent. Unfortunately, many desirable agronomic varieties do not produce embryos capable of germination when published protocols are followed. Using one responsive and three recalcitrant cultivars, we examined the effect on embryogenesis of auxin, nitrogen, and carbon; explant source; and desiccation. All cultivars formed proembryonic masses on medium supplemented with either 2,4-D or picloram; picloram favored the growth of nonembryogenic callus. Twenty mm each of ammonium and nitrate promoted the best proembryo formation in all cultivars. Ammonium was essential for embryogenesis; replacing ammonium with proline, glutamine, asparagine, glycine, or casein hydrolysate resulted in poor or no proembryo formation. More proembryos formed on medium supplemented with sucrose than with glucose, fructose, or maltose. Leaf discs from the first fully expanded leaf produced more embryos than younger leaves for all cultivars; discs taken from the lamina produced more embryos than discs including portions of the midrib. Proembryos matured and germinated only after at least 3 weeks on medium containing 5% w/v polyethylene glycol 8000, greater than 3.3 mm myo-inositol, and 1 or 10 μm abscisic acid. More whole plants were obtained from the responsive cultivar Jewel than from the recalcitrant genotypes.
M.A. Norton and D.R. LaBonte
D.W. Miano, D. R. LaBonte and C. A. Clark
Sweetpotato is an important staple food crop in Sub-Saharan Africa, with production being concentrated in East Africa, particularly around Lake Victoria. Productivity of the crop is greatly constrained by viral diseases. Four main viruses have consistently been detected from various surveys done in the region viz. sweet potato feathery mottle virus (SPFMV), sweet potato chlorotic stunt virus (SPCSV), sweet potato mild mottle virus (SPMMV), and sweet potato chlorotic fleck virus (SPCFV). Sweet potato caulimo-like virus (SPCaLV), sweet potato latent virus (SPLV), and cucumber mosaic virus (CMV) have also been detected though only in isolated cases. The most severe symptoms have been caused by co-infection with SPCSV and SPFMV, resulting in the synergistic Sweet potato virus disease (SPVD). Yield reductions due to virus infections have been estimated to be >90% in very severe cases. Virus detection has mainly been limited to the use of serological methods. Some plants have been observed with symptoms resembling those caused by viruses, but do not react with available antisera, indicating that the plants could be infected with viruses that have not been described, or not tested in the region. Use of other detection techniques such as PCR may result in identification of more viruses in the region. This report gives a summary of our research efforts towards detection of other viruses present in the region, and identification of resistant germplasm.
D.W. Miano, D. R. LaBonte and C. A. Clark
Sweet potato virus disease (SPVD) is a major constraint to sweetpotato production in East Africa. The disease is a result of co-infection with sweet potato feathery mottle virus (SPFMV) and sweet potato chlorotic stunt virus (SPCSV). Some local sweetpotato genotypes have been reported to recover from, or have localized distribution of SPVD, suggesting that the disease is not fully systemic. This has led to the suggestion that uninfected cuttings may be obtained from previously infected plants. Experiments were set to determine the possibility of obtaining cuttings long enough for propagation that are free from virus infection. This would form a basis for recommending to the local small-holder farmers of a way to reduce losses due to the disease. Field grown sweetpotato vines were cut into three pieces (15, 15 to 30, and >30 cm from the apex) and tested for SPCSV and SPFMV. Nine genotypes were selected from a group of 21 local clones and used for this study. The two viruses were equally present in all the three sections of infected vines, indicating that it is not easy to obtain a virus free cutting for field propagation from an infected vine.
M. Mcharo, D. LaBonte, R.O.M. Mwanga and A. Kriegner
Molecular markers linked to resistance to sweetpotato chlorotic stunt closterovirus [SPCSV (genus Crinivirus, family Closteroviridae)] and sweetpotato feathery mottle virus [SPFMV (genus Potyvirus, family Potyviridae)] were selected using quantitative trait loci (QTL) analysis, discriminant analysis and logistic regression. Eighty-seven F1 sweetpotato [Ipomoea batatas (L.) Lam.] genotypes from a cross of `Tanzania' and `Wagabolige' landraces were used to generate DNA marker profiles for this study. Forty-five of the clones were resistant to SPCSV while 37 were resistant to SPFMV. A combination of 232 amplified fragment length polymorphism (AFLP) markers and 37 random amplified polymorphic DNA (RAPD) markers obtained were analyzed to determine the most informative markers. All three statistical procedures revealed that AFLP marker e41m33.a contributed the greatest variation in SPCSV resistance and RAPD marker S13.1130 accounted for most of the variation in SPFMV resistance. The power of discriminant and logistic analyses is that you do not need a parent-progeny population. An evaluation of these two models indicated a classification and prediction accuracy rates of 96% with as few as four markers in a model. Both multivariate techniques identified one important discriminatory marker (e44m41.j) for SPCSV and two markers (e41m37.a and e44m36.d) for SPFMV that were not identified by QTL analysis.
Roberto A. Rivas, P.T. Evans and D.R. LaBonte
Methodology is presented for organogenesis of `Beauregard sweetpotato, a cultivar released in 1987 that is rapidly increasing in commercial use in the U.S. Regeneration of `Beauregard' sweetpotato plantlets was obtained when complete leaves and 10 mm internode explants were cultured in liquid and solid media respectively over a period of 8 weeks. Leaves in liquid Murashige and Skoog medium containing 2 mg/l of IAA placed on a shaker under dark conditions produced white callus at the cut end of the petiole and roots underneath the callus in 4 weeks. Leaves were subsequently transferred to MS medium containing 500 mg/l of Chlorocholine chloride (CCC) and 0, 1, 5 and 10 mg/l of BAP for 4 more weeks. Shoots were regenerated from callus using 1 mg/l of BAP.
The effect of NAA auxin and various concentrations of the cytokinin Thidiazuron on internodes was examined under 16 hr. light and 8 hr dark photoperiod using MS solid medium. Explants on 0.05 mg/l NAA alone produced roots and shoots. The most plantlets however, were regenerated using 0.05 mg/l of NAA and 0.01 mg/l Thidiazuron. Regeneration of plants from leaves and internodes may be a useful system for a clean and rapid propagation of `Beauregard' sweetpotato.
F.A. Buffone, D.R. LaBonte and C.A. Clark
Chlorotic leaf distortion is a common disease of sweetpotato caused by Fusarium lateritium. This fungus is unique among Fusarium species in that it grows epiphytically on leaves and shoot tips of sweetpotato. Fusarium lateritium mycelia appear as white masses on leaves, and this fungus can cause chlorosis under periods of bright sunlight. When environmental conditions are not favorable for growth, this organism is not readily observed on sweetpotato. The objective of this research was to see if DNA of F. lateritium is amplified using PCR techniques during amplification of sweetpotato DNA. Our results show cTAB extracts of sweetpotato inoculated with F. lateritium have additional bands not present in a control free of F. lateritium. Furthermore, these bands correspond to banding patterns obtained from the F. lateritium isolate DNA when amplified alone. Researchers who use sweetpotato tissue in PCR-based research, e.g., phylogenetic research, should be aware of these amplified products. This situation is further compounded because numerous F. lateritium biotypes are present in the environment.
C.D. Kokkinos, C.A. Clark, C.E. McGregor and D.R. LaBonte
Sweet potato virus disease (SPVD) is the most devastating disease of sweetpotato [Ipomoea batatas (L.) Lam.] globally. It is caused by the co-infection of plants with a potyvirus, sweet potato feathery mottle virus (SPFMV), and a crinivirus, sweet potato chlorotic stunt virus (SPCSV). In this study we report the use of cDNA microarrays, containing 2765 features from sweetpotato leaf and storage root libraries, in an effort to assess the effect of this disease and its individual viral components on the gene expression profile of I. batatas cv. Beauregard. Expression analysis revealed that the number of differentially expressed genes (P < 0.05) in plants infected with SPFMV alone and SPCSV alone compared to virus-tested (VT) plants was only 3 and 14, respectively. However, these findings are in contrast with SPVD-affected plants where more than 200 genes were found to be differentially expressed. SPVD-responsive genes are involved in a variety of cellular processes including several that were identified as pathogenesis- or stress-induced.
A.Q. Villordon, C.A. Clark, R.A. Valverde, R.L. Jarret and D.R. LaBonte
Previous work by our group has detected the presence of a heterogeneous population of Ty1-copia-like reverse transcriptase retrotransposon sequences in the sweetpotato genome. Recently, we detected the presence of putatively active Ty1-copia-like reverse transcriptase sequences from a virus-infected `Beauregard' sweetpotato clone. In the current study, we report the differential detection of putatively stress-activated sequences in clones from seedling 91-189. The clones were infected with different combinations of virus isolates followed by extraction of leaf RNA samples at three sampling dates (weeks 2, 4, and 6) after inoculation. After repeated DNAse treatments to eliminate contaminating DNA, the RNA samples were subjected to first strand cDNA synthesis using random decamer primers followed by PCR analysis utilizing Ty1-copia reverse transcriptase-specific primers. Through this approach, we detected amplified fragments within the expected size range (280-300 bp) from clones infected with isolates of sweetpotato leaf curl (SPLC) and feathery mottle viruses (FMV) (week 2 and 6) and FMV (week 4). We were unable to detect PCR products from the noninfected clones or the other infected samples. The data suggests that specific viruses may be involved in the expression of these Ty1-copia-related reverse transcriptase sequences. It also appears that sampling at various dates is necessary to detect putative activity over time. This preliminary information is essential before proceeding to the construction and screening of cDNA libraries to isolate and fully characterize the putatively active sweetpotato Ty1-copia-like retrotransposon sequences. Through the partial or complete characterization of sweetpotato Ty1-copia elements, sequences that correspond to cis-regulatory element(s) can be identified and further studied for their roles in responding to specific stress factors.
A. Villordon, S. Gichuki, H. Kulembeka, S.C. Jeremiah and D. Labonte
One of the secondary centers of genetic diversity for the sweetpotato [Ipomoea batatas (L.) Lam.] is located in Africa. We have developed a geo-referenced database of sweetpotato accessions for Tanzania and Kenya that is accessible by stakeholders and other users. Public domain base maps and other files were used to generate the underlying GIS components. DIVA-GIS was used to convert existing spreadsheet-based accession and passport data into GIS-compliant files. ALOV Map, a public domain Java application for publishing vector and raster maps, was used to provide the framework for a web-accessible GIS database. This demonstrates that the availability of publicly available software requiring minimal or flexible licensing costs provide a cost-effective alternative to institutions that are considering developing GIS databases as well as enabling web accessibility to such resources. DIVA-GIS was also used to predict potential distribution of sweetpotato germplasm in Sub-Saharan Africa using the built-in ecological niche modelling tool. We describe procedures, software, and other applications that we used to develop a publicly accessible web interface to a GIS database of sweetpotato germplasm collections in Kenya and Tanzania.
M.I. Buteler, D.R. LaBonte, R.L. Jarret and R.E. Macchiavelli
Using codominant molecular markers (microsatellites) for paternity identification was investigated in hexaploid sweetpotato [Ipomoea batatas (L.) Lam.]. Two experimental populations (CIP and LAES), each consisting of progeny of known parentage, were scored for the presence or absence of alleles segregating at IB-316 and IB-318 microsatellite loci. Paternity was assessed using paternity exclusion and the most-likely parent methods. In the former, paternity is assigned based on the identification of incompatible parent-progeny marker data. In contrast, the latter method incorporates paternity exclusion and a log-likelihood or LOD score that weighs progeny allelic patterns as to the likelihood that they could have come from a given paternal parent. The number of correctly allocated progeny differed for the methods. Paternity exclusion correctly allocated 7% and 25% of the progeny in the LAES and CIP populations, respectively. The most-likely parent method correctly allocated 23% and 88% of the progeny in the LAES and CIP populations, respectively. The greater misassignments in the LAES population were attributed to low allelic diversity at the LAES IB-318 locus and a larger parental population. This study demonstrates the feasibility of identifying paternity in sweetpotato using a minimal number of loci.