Optimization of parameters influencing biolistic transformation is a crucial stage towards repeatable transformation of common beans. However, there has been no published study on such optimization of this crop species in a helium particle delivery system (BioRad). Using an intron-containing β-glucuronidase (GUS) gene as a reporter, we optimized several critical parameters of biolistic PDS-1000/He delivery system for common bean transformation. The target explant tissues included cotyledons, zygotic embryos, and meristemic shoot tips suitable for organogenesis. Thus, pretreatment of target tissues with osmotic medium containing 0.15–0.25 m mannitol and 0.15–0.25 m sorbitol, positioning of target tissues in 4 cm microcarrier flying distance, the use of 1.6-μm gold particle and high concentration of coating DNA, and bombardment of young immature tissues twice at 2000 psi, etc., significantly increased transformation rate and achieved the best coverage and penetration of the meristemic areas involved in direct shoot organogenesis.
Zhanyuan Zhang, A. Mitra and D.P. Coyne
Zhanyuan Zhang, A. Mitra and D.P. Coyne
Factors influencing Agrobacterium–mediated DNA transfer of P. vulgaris were examined using an intron-containing β-glucuronidase (GUS) gene as a reporter system. Tissue culture procedures used were based on direct shoot organogenesis. Two A. tumefaciens strains, A2760 and EHA105, were used with more emphasis on the former due to its overall higher transformation rate. Ten bean entries including breeding lines and cultivars from both Meso-American and Andean origins were compared for compatibility with the two bacterial strains under different pre- and coculture conditions. Pinto `Othello' was extensively used in testing different transformation conditions. Factors found to have significant effects on transformation rate included Agrobacterium-host interactions, explant maturity, preculture and cocultivation conditions, as well as selection schemes, based on transient expression. Some factors, such as the effect of explant maturity and dark preconditioning of explants on gene transfer, have not been reported before. The best transformation conditions included the use of susceptible genotypes and mature explants, preconditioning of explants in darkness, followed by a maximum cocultivation period in the presence of cytokinin, and the use of high selection pressure.
Zhanyuan Zhang, D.P. Coyne and A. Mitra
Gene transfer can provide plants with a novel source of disease resistance. Two different antibacterial peptides, Shiva-1 and lactoferrin, were tested in vitro for antibacterial activity. The former is from cecropin B in insects, and the latter from human or mammal fluids such as milk. Both peptides exhibited high antibacterial activity against all tested gram-negative phytopathogenic bacterial strains. Lactoferrin was more lethal than Shiva-1. A particular lactoferrin domain showed a much higher activity against bacterial strains. A gene encoding lactoferrin was then transferred to Nicotinia tabacum L. xanthi-nc to evaluate the gene expression using Agrobacterium. Stable transformation was confirmed by Southern, Northern, and Western blot analysis. Delayed wilting of the transgenic plants inoculated with Pseudomonas solanacearum was observed. A significant positive relationship between the gene expression levels and resistance was also found by either Northern or Western blotting. Biolistic transformation using a gene gun is currently underway to transfer this novel gene to common beans.
D.P. Zhang, M. Ghislain, A. Golmirzaie and J.C. Cervantes
Detecting inter- and intra-varietal variation is essential for the management of a plant germplasm bank. The sensitivity and efficiency of randomly amplified polymorphic DNA (RAPD) for cultivar identification and somaclonal mutation in sweetpotato were evaluated. RAPD demonstrated a highly significant inter-varietal variation. Every one of the 23 tested cultivars can be identified with a RAPD profile generated by a single primer. Suspected duplicates that are morphologically indistinguishable can be unambiguously verified with a combination of three decamers. No intra-varietal variation was found using RAPD. Clones of `Jewel' and `Beauregard' collected from different sources all have the same RAPD profiles. Moreover, with 150 markers, the transgenic `Chogoku' sweetpotato cannot be differentiated from its untransformed counterparts, even though the transgenic plant shows significant morphological changes. These results demonstrate that RAPD is a sensitive and efficient tool for identifying cultivar duplicates, but it is not efficient for detecting intra-clonal variation or somaclonal mutation in sweetpotato.
A. Golmirzaie, D.P. Zhang, L. Nopo, C.A. Newell, A. Vera and F. Cisneros
Euscepes postfasciatus is one of the most important sweetpotato pests in the South Pacific, Caribbean basin, and some countries of Central and South America. Development of host resistance will greatly improve the effects of integrated pest management (IPM) for this pest. Ten transgenic clones of `Jewel' sweetpotato with cowpea trypsin inhibitors and snowdrop lectin, developed by Axis Agri. Genetics, Ltd., were assayed for weevil resistance using a no-choice bioassay. A replicated experiment was conducted in the screenhouse. Five storage roots from each clone were infested with five pairs of adults. Non-transformed `Jewel' was used as a check. Resistance was assessed 60 days after infestation by estimating the percentage of internal damage and the weevil population in the storage roots. A five-grade damage index was recorded. The experiment was repeated twice. Significant enhancement of resistance was found in the transgenic clones. Clone CTI-13 with cowpea trypsin inhibitor and clone PCG-7 with both cowpea trypsin inhibitor and snowdrop lectin demonstrated moderate resistance to E. postfasciatus, whereas the non-transformed `Jewel' was susceptible. This result shows that resistance to Euscepes postfasciatus can be achieved through genetic transformation.
R.O.M. Mwanga, A. Kriegner, J.C. Cervantes-Flores, D.P. Zhang, J.W. Moyer and G.C. Yencho
When sweetpotato chlorotic stunt crinivirus (SPCSV) and sweetpotato feathery mottle potyvirus (SPFMV) infect sweetpotato [Ipomoea batatas (L.) Lam.], they interact synergistically and cause sweetpotato virus disease (SPVD), a major constraint to food productivity in east Africa. The genetic basis of resistance to these diseases was investigated in 15 sweetpotato diallel families (1352 genotypes) in Uganda, and in two families of the same diallel at the International Potato Center (CIP), Lima, Peru. Graft inoculation with SPCSV and SPFMV resulted in severe SPVD symptoms in all the families in Uganda. The distribution of SPVD scores was skewed toward highly susceptible categories (SPVD scores 4 and 5), eliminating almost all the resistant genotypes (scores 1 and 2). Likewise, when two promising diallel families (`Tanzania' × `Bikilamaliya' and `Tanzania' × `Wagabolige') were graft inoculated with SPCSV and SPFMV at CIP, severe SPVD was observed in most of the progenies. Individual inoculation of these two families with SPCSV or SPFMV, and Mendelian segregation analysis for resistant vs. susceptible categories led us to hypothesize that resistance to SPCSV and SPFMV was conditioned by two separate recessive genes inherited in a hexasomic or tetradisomic manner. Subsequent molecular marker studies yielded two genetic markers associated with resistance to SPCSV and SPFMV. The AFLP and RAPD markers linked to SPCSV and SPFMV resistance explained 70% and 72% of the variation in resistance, respectively. We propose naming these genes as spcsv1 and spfmv1. Our results also suggest that, in the presence of both of these viruses, additional genes mediate oligogenic or multigenic horizontal (quantitative) effects in the progenies studied for resistance to SPVD.