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.
H.M. Ariyarathne and D.P. Coyne
Halo blight is one of the most important bacterial diseases of common beans (Phaseolus vulgaris L.). It is serious under moderate temperature and high humidity conditions. The disease is caused by a seed-borne bacterium, Pseudomonas syringae pv. phaseolicola (Burkh.) Dowson (Psp). The inheritance of leaf reactions to Psp, flower, and stem color was studied using greenhouse-grown 109 F9 recombinant inbred lines (RI) from the P. vulgaris cross BelNeb 1 [resistant (R)] (USDA/NE) × A 55 [susceptible (S)] (CIAT). Two Psp strains, HB16 (NE) and 83-Sc2A (NE), were inoculated using the water-soaking method. A segregation ratio of 1 R:1 S RI lines were observed for disease reactions in leaves for both strains indicating major gene control. The presence of recombinants for SR, RS to the strains indicated that different genes were involved. Stem (SC) and flower (FC) color traits were each determined by two major genes. Linkages were found for reactions to the two Psp strains and also between FC and SC. No linkages were observed from FC and also SC with reactions to Psp strains.
E. Mergenthaler, Gy. Bisztray and D.P. Coyne
As ancestors of higher plants, mosses offer advantages as simple model organisms in studying complex processes. The moss Physcomitrella patens became a powerful model system in the last few years (Cove and Knight, 1993). Adaptation of PEG-mediated DNA uptake procedure has permitted the establishment of efficient molecular genetic approaches. To study possible effects of a Type I phytochrome, the potato phyA gene was introduced into the moss P. patens. Stabile transformants exhibited a range of similar phenotypes (Schaefer et al., 1991). The aim was to differentiate the wild type from the transgenic moss plants with simple, quick measurements providing data suitable for analyzing offspring populations. Ten different morphological and biochemical methods were used to investigate the phenotype in order to choose the best phenotypical category to indicate the presence and the effect of the phytochrome transgene. Two selected strains were used with the most and the least intensive phenotypical features (3*, 29), along with their selfed progenies, as well as progenies from crosses with the nicotinic-acid auxotrophic mutant. The best methods to differentiate between wild type and transgenic plants were the statistical analysis of the number of gametophores, photometric measurement of pigment contents and composition under different light conditions, color evaluation by PC-based vision system, and visual observation of morphogenetic changes. Our investigations support that the potato phytochrome transgene has a pleiotropic effect in the moss P patens. The methods used would be applicable for the characterization of mosses with different transgenes.
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.
Mohamed F. Mohamed, P. E. Read and D. P. Coyne
Regeneration in vitro from the embryonic axis in Phaseolus sp. has not been reported. Two embryo sizes, 0.3-0.4 mm and 0.6-0.7 mm long at 10-12 and 21 days after pollination, respectively, were excised from 4 P. vulgaris (P.v.) and 2 P. acutifolius (P.a.) genotypes. The embryonic leaves and radicale were removed, and 0.1-0.2 mm of the embryonic axis was cultured on Gamborg's B5 medium with 0, 5, 10 and 20μ MBA. The cultures were incubated in the dark at 25°C for 2 weeks followed by 1 week in continuous cool white light (25μ MS-1m2) before transferring to the second medium (0, 2μ MBA and 2μ MBA + 4μ MGA3). The tissues from the larger embryos initiated a single shoot without PGR in 30% of 1 P.v. explants and 30-60% in 2 P.a. The other 3 P.v. formed roots only. Multiple shoots were initiated in all P.v. (15-60%) and in 2 P.a. (60 and 70%) with 5 or 10μ MBA. The tissues from the smaller embryos had single shoots for all genotypes (30-60%) without PGR. Multiple shoots were initiated in 50-80% and 75-90% of the explants from P.v. and P.a., respectively, with 5 or 10μ MBA. Excess callus formed with 20μ MBA and regeneration decreased. After 3 weeks on the second medium, 6-8 shoot s/P. v. and up to 15-20 shoots/Pa. explants were observed.
D. P. Coyne, E. Arnaud-Santana, J. Beaver and H. Zaiter
Some dry bean lines (L) Phaseolus vulgaris resistant (R) (compatible reaction) to Xanthomonas campestris var. phaseoli (Xpc) developed in the temperate zone express susceptibility (S) in the field in the tropics and tropical lines (S) express moderate R in the temperate zone. There is only limited information on the influence of P on Xpc reaction in dry beans. Experiments were conducted in growth chambers (GC) and in the field (NE, Dominican Republic, Puerto Rico) to investigate the influence of P and P × temperature (T) on the reaction of L to Xpc. A split-plot design was used with T as the main-plots and P and L as sub-plots in the GC experiment and with P as main-plots and L as sub-plots in the field experiments. The disease reactions were more severe on L under short P and under higher T than under shorter P and lower T. No interactions were detected among these treatments. PC-50 showed moderate R, delayed flowering, flower bud abortion, and increased branching under long P (field, NE). These results have implications for breeders in the evaluation of field R of L from different latitudes and for their value in breeding.
S.O. Park, J.M. Bokosi and D.P. Coyne
Plant growth habit is an important trait. Our objective was to identify RAPD markers linked to major gene for indeterminate growth habit using bulked segregant analysis in an F2 population from a bean cross Chichara (indeterminate growth habit × PC-50 (determinate growth habit). A total of 132 RAPD primers (600 RAPD primer screened) showed polymorphisms between bulked DNA derived from indeterminate and determinate plants. All markers showed coupling linkage with indeterminate growth habit. RAPD markers of A-8, A-17, C-7, C-15, D-4, D-5, F-6, F-16, G-9, H-3, H-20, and I-7 were 2.2 cM distant from the gene for indeterminate growth habit. Markers of B-7, B-16, B-17, C-8, E-1, F-1, F-20 and H-l9 primers were 4.6 cM distant from the gene for indeterminate growth habit.
S.O. Park, A. Dursun, D.P. Coyne and G. Jung
Common bacterial blight (CBB), incited by Xanthomonas campestris pv. phaseoli (Xcp), an important disease in common bean (Phaseolus vulgaris L.) Tepary bean (P. acutifolius A. Gray) is of interest to bean breeders because of resistance to CBB. Our objective was to identify RAPD markers linked to major genes for CBB resistance using bulked segregant analysis in an F2 population from a tepary bean cross CIAT640005 (R) X Nebr#4B (S). A total of 57 RAPD primers (602 RAPD primers screened) showed polymorphisms between bulked DNA derived from R and S CBB plants. All markers showed coupling linkage with CBB resistance. A good fit to a 3:1 ratio of bands for presence and absence using 11 RAPD primers was observed in 77 F2 plants. Markers of U-15 and L-7 primers were 2.4 cM distant from the gene for resistance to Xcp strain LB-2. RAPD markers of U-10, U-20, S-12, Y-4, F-13, P-6, Q-1, and Q-ll primers were 2.4 cM distant from the gene for resistance to Xcp strain SC-4A. RAPD markers of IJ-15 and L-7 primers were 8.4 cM distant from the gene for resistance to Xcp strain EKl l. The tepary RAPD linkage group includes three molecular markers and three genes for resistance to Xcp strains EK-l l, LB-2, and SC-4A and spans a length of 19.2 cM. This data supports the presence of Xcp races.