Ten common bean (Phaseolus vulgaris L.) lines—including cultivars, breeding lines, and one wild line—were evaluated for susceptibility to Agrobacterium tumefaciens strain C58 by stab-inoculating intact shoot tips of germinating seeds. Significant differences for tumor frequency and size were found on the resulting 3-week-old seedlings. UW 325, a wild bean, had the highest rate of tumorigenesis; `Olathe', a dry bean cultivar, had the lowest. Uninoculated excised shoot tips cultured in media with BA or BA plus NAA exhibited differences in phytohormone sensitivity, as evidenced by callusing and root initiation. The cultivar Montcalm seemed to be highly sensitive, while `Olathe' was relatively insensitive. Fluorometric GUS assays of shoot tips from germinating seeds inoculated with the disarmed GUS-containing A. tumefaciens strain C58C1(pGV3850/pKIWI105) showed that UW 325 had the highest level of GUS activity. `Montcalm' had a high rate of tumorigenesis but a low level of GUS activity; this anomaly was attributed to its high phytohormone sensitivity. The use of the virulence-inducing compound acetosyringone in the inoculum culture medium did not alter genotypic differences (ranks) in susceptibility. Histochemical GUS assays of inoculated UW 325 shoot tips showed that 60% of the apexes exhibited one or more transformation events. Chemical names used: β-glucuronidase (GUS); α-naphthaleneacetic acid (NAA); N-(phenylmethyl)-1H-purin-6-amine (BA).
Mark E. Lewis and Fred A. Bliss
Sandra L. Uratsu, Hamid Ahmadi, Royce S. Bringhurst and Abhaya M. Dandekar
Several strains of Agrobacterium tumefaciens and A. rhizogenes were shown to form tumors on runners of the diploid strawberry species Fragaria vesca L. Tumors, weighing from 0.1 to 8.3 mg, appeared from 2 to 4.5 weeks after infection. The majority of tumors tested for opine synthesis by high-voltage paper electrophoresis analysis showed positive results. These results demonstrate that diploid strawberry plants are susceptible to infection with Agrobacterium and that there are differences in the relative virulence of Agrobacterium strains.
Ana Cristina M. Brasileiro, Francisco J. Lima Aragão, Sílvia Rossi, Diva Maria A. Dusi, Leila M. Gomes Barros and Elíbio L. Rech
To develop an efficient protocol for Agrobacterium-mediated transformation of common bean (Phaseolus vulgaris L.) and tepary bean (P. acutifolius A. Gray), we have tested the susceptibility of six genotypes to eight Agrobacterium tumefaciens and two A. rhizogenes strains. The virulence of the Agrobacterium strains was shown to be genotype dependent. In general, the tumors observed on common bean cultivars were larger than those observed on tepary bean cultivars. The A. tumefaciens AT8196 and Ach5 strains and the A. rhizogenes 8196 strain induced the best responses in all genotypes tested. Polymerase chain reaction (PCR) analysis confirmed the presence of T-DNA in tumors derived from inoculation with three A. tumefaciens strains in common beans. Apical meristems of P. vulgaris cv. Jalo were bombarded with tungsten microprojectiles and then inoculated with an A. tumefaciens wild-type strain (Ach5). One month later, the explants showed a high frequency of tumor formation (50% to 70%). Similarly, when bombarded meristems were inoculated with an A. tumefaciens disarmed strain (LBA4404/p35SGUSINT), 44% of them showed substantial sectors of GUS activity, suggesting the expression of introduced gene. The bombardment/Agrobacterium system appears to be a promising method to stably transform bean through the regeneration of plants directly from transformed apical meristems.
Antonio Figueira, Kelson Kodama, Isabela Sathler, Eva Mamani and Siu Mui Tsai
Resistance genes (QTLs and major genes) to various pathogens tend to occur in clusters at specific locations in plant genomes. In common bean (Phaseolus vulgaris), common genomic regions associated with host response to the bacterial pathogen Xanthomonas campestris pv. phaseoli and to the symbiont Rhizobium have been identified. Agrobacterium tumefaciens causes crown gall disease in common bean, and shares similar gene activation mechanisms and host recognition signals with Rhizobium. Genotypic differences in tumorigenesis have been observed and could restrict Agrobacterium-mediated bean genetic transformation. The objectives of this research were: 1) to identify wild Agrobacterium strains inducing contrasting response in bean genotypes; and 2) to identify genomic regions in a core linkage map associated with host response to Agrobacterium infection, in comparison with the position of other symbiont or pathogen resistance genes. Among 10 wild A. tumefaciens strains tested under controlled inoculations of 1-week-old seedlings, Ach5, R10, and mainly Chry5 were virulent to the genotypes tested. The genotype BAT93 was susceptible to Chry5, even at low inoculum concentration, in contrast with JaloEEP558. Increasing levels of N enhanced susceptibility to Chry5, R10 and Ach5. Fifty recombinat inbred lines of BAT93 × JaloEEP558 were inoculated with Chry5 and segregated for tumor formation, with 21 lines not forming tumors. Analysis of variance identified 25 markers in five linkage groups of the core linkage map, significantly associated with Agrobacterium resistance, sharing the same location with QTLs identified for other host-bacteria interactions. (Financed by FAPESP 97/12066-1).
Mark H. Brand and Richard Kiyomoto
Tissue proliferation (TP) of Rhododendron sp. is characterized by basal tumors that often develop into numerous dwarf shoots. Growers need to know if the TP condition will recur in plants grown from normal-appearing cuttings collected from plants with TP tumors. Stem cuttings of seven cultivars were collected from stock plants with TP [TP(+)] and without TP [TP(–)] and rooted. Plants were grown in containers outdoors for 2 years and were then evaluated for tumor formation and other TP-related morphological symptoms. Shoots of TP(+) plants were either similar in length to shoots of TP(–) plants, or were shorter, as was the case for `Boule de Neige', `Catawbiense Album', and `Montego'. Plants grown from TP(+) cuttings of all cultivars had more leaves per growth flush than did plants grown from TP(–) cuttings. `Holden', `Montego', and `Scintillation' TP(+) leaves were narrower than leaves from TP(–) shoots and had greater length: width ratios. Leaves of TP(+) `Montego' and `Scintillation' plants were shorter and smaller than leaves from their TP(–) counterparts. Tumors were not observed on any propagated plants, regardless of the TP status of cutting stock plants. To further test the influence of age and TP status of source plants used for cutting propagation, `Montego' plants were grown from cuttings collected from the following sources: 1) in vitro shoot cultures; 2) 3-year-old plants with TP; 3) 6-year-old plants with TP; and 4) TP(–) plants. Cuttings from TP(+) micropropagated plants less than 3 years old were more likely to develop tumors than were cuttings from older plants. Eighty-three percent of plants from microcuttings and 74% of plants from cuttings of 3-year-old TP(+) plants formed tumors, whereas no plants grown from 6-year-old TP(+) or TP(–) cuttings did so. Large tumors that surrounded half or more of the stem were more likely to develop on plants grown from microcuttings than on plants grown from the next youngest, 3-year-old TP(+), stock plants. Growers must be aware that cuttings from TP(+) plants may produce plants that exhibit morphological and growth abnormalities, possibly even including tumor redevelopment.
Christopher C. Dickinson, Alexandra J. Weisberg and John G. Jelesko
gene transformation capability of A. tumefaciens was reengineered to replace the tumor formation regions on the Ti plasmid (i.e., disarmed binary Ti plasmids) with recombinant “genes of interest.” The potential utility of Agrobacterium
Fengge Hao, Lirong Wang, Ke Cao, Xinwei Wang, Weichao Fang, Gengrui Zhu and Changwen Chen
treated with sterilized distilled water did not result in tumor formation (data not shown). To increase the frequency of inoculation replication, the seedlings were inoculated for the second time at 30 d after the first inoculation. Except the two