Genotypic Variability of Soybean Response to Agrobacterium Strains Harboring the Ti or Ri Plasmids

We have examined the response of the hormone-resistant mutants axrl and axr2 of Arabidopsis thaliana to inoculation by Agrobacterium tumefaciens and Agrobacterium rhizogenes. Our results indicate that recessive mutations in the axrl gene affect the frequency of tumor formation after inoculation with either Agrobacterium strain. In addition, tumors produced on axrl plants were smaller than those growing on wild-type plants. These results indicate that the product of the AXR1 gene is important for both crown gall and hairy root tumor formation. In contrast, the dominant axr2 mutation has a more severe effect on the development of crown gall tumors than on hairy root tumors. Crown gall tumors produced on axr2 plants had a different morphology than wild-type tumors and did not grow when they were removed from the explant. In contrast, a large number of hairy root tumors were produced on wild-type and axr2 plants, and both types of tumors grew when they were removed from the explant. Like the roots of axr2 plants, roots produced on axr2 explants lacked root hairs.

Section: Discussionmentioning

confidence: 99%

Hormone-Resistant Mutants of Arabidopsis Have an Attenuated Response to Agrobacterium Strains

Lincoln¹,

Turner²,

Estelle³

1992

“…Crown gall tumorigenesis has been measured in many plants using various tissues, including leaves and stems of intact plants (for example, 5,17,22,28), and explants ofroots (13), tubers (1), and cotyledons (15,27). Lacking is an assay that is based on infection of host plants at the natural site of infection of Agrobacterium tumefaciens, the root.…”

Section: Discussionmentioning

confidence: 99%

“…Genetic variation in susceptibility to crown gall has been exploited to analyze resistance genes in grapevine, an economically important host of A. tumefaciens (31 (24), alfalfa (21), kalanchoe (3), castor bean (7), sunflower (4), soybean (15,27), and squash (29,33). The results indicate that genetic variation in response to A. tumefaciens is readily detectable in natural populations.…”

Section: Discussionmentioning

confidence: 99%

Use of a Root Tumorigenesis Assay to Detect Genotypic Variation in Susceptibility of Thirty-four Cultivars of Pisum sativum to Crown Gall

Hawes¹,

Robbs²,

Pueppke³

1989

We developed a quantitative assay to measure tumorigenesis on roots and root crowns, the natural sites of Agrobacterium tumefaciens infection. Efficiency of tumor formation and tumor weight on seedlings of Pisum sativum 'Little Marvel' were directly proportional to the logarithm of inoculum concentration. Depth of wounding prior to inoculation also significantly influenced tumor weight but not efficiency. Mean weight of tumors that developed in response to inoculation with strain B6 varied significantly among 34 different commercial cultivars. Tumors on the most susceptible cultivar, Target, were more than tenfold heavier than those formed on the least susceptible cultivar, Sweet Snap. Efficiency of tumorigenesis on 'Sweet Snap' was also relatively low: only 64% of inoculated seedlings developed tumors compared with 89 to 100% efficiencies for all other cultivars. evaluate resistance and susceptibility in Pisum sativum L., a well characterized diploid species that is a known host of A. tumefaciens (16). To initiate studies of crown gall resistance in pea, we established a reproducible quantitative assay for tumorigenesis on roots of intact plants and used it to survey genotypic variation among 34 commercial cultivars of pea. MATERIALS AND METHODS Bacterial Strains and CultureSingle colony cultures of bacterial strains were maintained at -70C in 50:50 (v/v) glycerol:yeast extract-mannitol (YEM) medium. Strain B6 was a gift from J. A. Lippincott (Northwestern University). For assays, cultures were grown overnight on YEM solidified with 0.7% agar and were suspended in water. Inoculum concentrations were estimated turbidimetrically and confirmed by dilution plating.Agrobacterium tumefaciens is a soil-borne bacterial pathogen that causes crown gall on most dicots and some gymnosperms and monocots (5). Although most parts ofmany plants are susceptible to experimental inoculation, A. tumefaciens normally infects through wounds in roots and at the rootshoot interface, the crown (18). Crown gall is among the major disease problems in fruit tree and ornamental nurseries and vineyards (25,26 Plant Inoculation and AssayPisum sativum L. seeds were surface-sterilized by consecutive 5-min immersions in 95% ethanol and 50% commercial bleach and then washed 4x in at least 50 mL of sterile water. Seeds were germinated on water agar overlaid with filter paper for 2 to 3 d or until the radicles were approximately 1 to 3 cm in length. Each seedling was wounded either by stabbing the root with a marked scalpel blade to a depth of 1 mm or by pushing the blade through the root to a depth of approximately 3 mm. Three wounds were made on each plant at approximately 5-mm intervals from the crown to the root tip. Seedlings were immersed into 5 mL of inoculum for 5 min and were then placed into moistened growth pouches (Northrup King, Minneapolis, MN), with 4 or 5 seedlings per pouch. Pouches were moistened daily with approximately 5 mL of water and were incubated at 250C day/230C night for 2 weeks.

“…The Agrobacterium-mediated gene transfer system would appear to have some advantages because soybean is a dicotyledonous plant species and thus is susceptible to Agrobacterium infection. However, infectivity tests show that not all Agrobacterium strains can readily infect soybean tissues (5,9,22). The Agrobacterium strains which have been 1212 found to be the most effective for the infection of soybean cultivars are those known as the nopaline-type, which encode for the synthesis of the novel amino acid nopaline (8).…”

mentioning

confidence: 99%

Transformation of Soybean (Glycine max) by Infecting Germinating Seeds with Agrobacterium tumefaciens

Chee¹,

Fober²,

Slightom³

1989

136

The transfer of genetic material into soybean tissue was accomplished by using an avirulent strain of Agrobacterlum tumefaciens which contained the binary vector pGA482. The method used for transformation requires no tissue culture steps as it involves the inoculation of the plumule, cotyledonary node, and adjacent cotyledon tissues of germinating seeds. The identification of neomycin phosphotransferase (NPT) II enzyme activity in the tissues of 16 (Ro) soybean plants indicated that the plant expressible Nos-NPT II gene, contained within the T-DNA region from pGA482, had been transferred at least into somatic tissues.Putative transformed Ro soybean plants were advanced to produce R1 plants which were also assayed for the presence of the transferred Nos-NPT II gene. The combined results of these assays indicated that about 0.7% of the surviving inoculated seeds yielded transformed tissues in the Ro plant, and that about 1/10 of these plants yielded transformed R1 plants. The presence of the Nos-NPT II gene in DNAs isolated from both Ro and R1 plant was demonstrated by using genomic blot hybridization and polymerase chain reaction methods. Integration of this gene into the soybean genome was demonstrated for three R1 soybean plants.