Agrobacterium tumefaciens was used to genetically transform sorghum. Immature embryos of a public (P898012) and a commercial line (PHI391) of sorghum were used as the target explants. The Agrobacterium strain used was LBA4404 carrying a `Super-binary' vector with a bar gene as a selectable marker for herbicide resistance in the plant cells. A series of parameter tests was used to establish a baseline for conditions to be used in stable transformation experiments. A number of different transformation conditions were tested and a total of 131 stably transformed events were produced from 6175 embryos in these two sorghum lines. Statistical analysis showed that the source of the embryos had a very significant impact on transformation efficiency, with field-grown embryos producing a higher transformation frequency than greenhouse-grown embryos. Southern blot analysis of DNA from leaf tissues of T0 plants confirmed the integration of the T-DNA into the sorghum genome. Mendelian segregation in the T1 generation was confirmed by herbicide resistance screening. This is the first report of successful use of Agrobacterium for production of stably transformed sorghum plants. The Agrobacterium method we used yields a higher frequency of stable transformation that other methods reported previously.

Agrobacterium-mediated sorghum transformation.

A high throughput genetic transformation system in maize has been developed with Agrobacterium tumefaciens mediated T-DNA delivery. With optimized conditions, stable callus transformation frequencies for Hi-II immature embryos averaged approximately 40%, with results in some experiments as high as 50%. The optimized conditions include N6 medium system for Agrobacterium inoculation, co-cultivation, resting and selection steps; no AgNo3 in the infection medium and adding AgNo3 in co-cultivation, resting and selection medium; Agrobacterium concentration at 0.5×109 c.f.u. ml−1 for bacterium inoculation; 100 mg l−1 carbenicillin used in the medium to eliminate Agrobacterium after inoculation; and 3 days for co-cultivation and 4 days for resting. A combination of all of these conditions resulted in establishing a high throughput transformation system. Over 500 T0 plants were regenerated and these plants were assayed by transgene expression and some of them were also analyzed by Southern hybridization. T1 plants were analyzed and transmission of transgenes to the T1 generation was verified. This represents a highly reproducible and reliable system for genetic transformation of maize Hi-II.

High throughput genetic transformation mediated by Agrobacterium tumefaciens in maize

Veterinary drugs in the environment and their toxicity to plants.

Sonication-assisted Agrobacterium-mediated transformation (SAAT) tremendously improves the efficiency of Agrobacterium infection by introducing large numbers of microwounds into the target plant tissue. Using immature cotyledons of soybean as explants, we evaluated the effects of the following parameters on transient β-glucuronidase (GUS) activity: cultivars, binary vectors, optical density of Agrobacterium during infection, duration of sonication treatment, co-culture conditions, length of explant preculture and addition of acetosyringone during co-culture. The extent of tissue disruption caused by sonication was also determined. The highest GUS expression was obtained when immature cotyledons were sonicated for 2 s in the presence of Agrobacterium (0.11 OD600nm) followed by co-cultivation with the abaxial side of the explant in contact with the culture medium for 3 days at 27°C. The addition of acetosyringone to the co-culture medium enhanced transient expression. No differences were observed when different cultivars or binary vectors were used. Cotyledons sonicated for 2 s had moderate tissue disruption, while the longer treatments resulted in more extensive damage.

Sonication-assisted Agrobacterium-mediated transformation of soybean immature cotyledons: optimization of transient expression.

Influence of the antibiotic timentin on plant regeneration compared to carbenicillin and cefotaxime in concentrations suitable for elimination of Agrobacterium tumefaciens

The effects of various antibiotics on the development of hypocotyls of Antirrhinum majus in tissue culture have been studied. The penicillins, carbenicillin and penicillin G, have been shown to stimulate callus growth, have little impact on shoot production and may stimulate root formation. The cephalosporins, cephotaxime and cephalosporin, have no effect on callus production and reduce shoot and root formation. HPLC, GC and GC-MS analyses have shown that concentrations of carbenicillin and penicillin G, commonly used in plant tissue culture, break down to give physiologically active levels of the auxin phenylacetic acid. This offers a mechanism for the stimulation of growth caused by these two antibiotics.

The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus.

The effects of varying the pH of the cocultivation medium, additons of vir-inducing phenolic compounds and the strains of wild-type agrobacteria on transformation rates of a number of different varieties of Antirrhinum majus were studied. In general, optimal transformation was found with strains C58 or A281 and was favoured by low pH and the inclusion of acetosyringone in the co-cultivation medium. However, maximal transformation of the least susceptible variety was achieved at high pH and in the presence of syringaldehyde. This demonstrates the need for the optimization of a wide range of culture conditions when working with new genotypes and offers a rational approach towards the development of Agrobacterium-mediated transformation of new species or varieties.

P. Holford

Papers

The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus.

Factors influencing the efficiency of T-DNA transfer during co-cultivation of Antirrhinum majus with Agrobacterium tumefaciens.