Marker-gene-free transgenic soybean plants were produced by isolating a developmentally regulated embryo-specific gene promoter, app1, from Arabidopsis and developing a self-activating gene excision system using the P1 bacteriophage Cre/loxP recombination system. To accomplish this, the Cre recombinase gene was placed under control of the app1 promoter and, together with a selectable marker gene (hygromycin phosphotransferase), were cloned between two loxP recombination sites. This entire sequence was then placed between a constitutive promoter and a coding region for either beta-glucuronidase (Gus) or glyphosate acetyltransferase (Gat). Gene excision would remove the entire sequence between the two loxP sites and bring the coding region to the constitutive promoter for expression. Using this system marker gene excision occurred in over 30% of the stable transgenic events as indicated by the activation of the gus reporter gene or the gat gene in separate experiments. Transgenic plants with 1 or 2 copies of a functional excision-activated gat transgene and without any marker gene were obtained in T0 or T1 generation. This demonstrates the feasibility of using developmentally controlled promoters to mediate marker excision in soybean.

No abstract

Randomly amplified polymorphic DNA (RAPD) profiles are currently being developed for Laccaria and Hydnangium species and Rhiwcfonia solani. The technique is increasingly being used to differentiate fungal isolates. As for the polymerase chain reaction (PCR) from which it was derived, the conditions necessary for reproducible RAPD products have received attention. However, in contrast to the PCR reaction, the technique relies on non-specific primers and as a consequence the reaction conditions are not necessarily as specific as they are in PCR. Compared with PCR products, RAPD fingerprints were therefore more sensitive to reaction and thennocycle conditions. RAPD products produced using 10 and 17-23 mer primers were visualized on ethidium bromide stained p~l~acrylarnide electrophoresis gels. Factorial experiments showed RAPD patterns were altered by changes in various reaction mixture components including the concentration of non-DNA impurities, the number and concentration of primers and the DNA polymerase enzyme type, source and concentration. These factors, particularly the enzyme source, reacted differently with the magnesium chloride concentration. Fluorescent dye labelled primers were used with internal lane standards in a DNA sequencing system to assess accurately the molecular weights and relative amounts of reaction products. Attachment of the fluorescent label appeared to favour the synthesis of some fragments compared with others on patterns visualized on ethidium bromide stained non-denaturing polyacrylamide gels. The temperature at which DNA was denatured in the first cycles altered the fingerprints. Reaction mixture temperatures of 9 4 ' or higher, compared with 91-93O, caused loss of visual yield of some products, particularly those greater than 500 base pairs, and increased the yield of others. Reproducibility of RAPD patterns, when the reaction mixture and temperature profile factors were varied, was facilitated by cross reference to fluorescently labelled bands separated with internal lane standards in a DNA sequencing system. Reproducibility of fingerprint patterns in a standard reaction mixture was achieved, with different thennocycle programmes and in different thennocyclers when the temperature profile was reproduced, suggesting that particular RAPD fingerprints may be reproduced in any laboratory provided the same set of reaction and thermocycle conditions are used. DNA fingerprinting is a technique which has been widely adopted in order to differentiate among organisms at the species and subspecies levels (Maclean ef al., 1993). Restriction fragment length polymorphism (RFLP) analysis has been used successfully to distinguish closely related fungi (Forster et al., 1987; Levy ef al., 1991). This technique is time consuming and requires large quantities of DNA and specific probes. Recently Williams ef al. Welsh &McClelland (1990) described a technique which makes use of single species of arbitrary primers to amplify DNA polymorphisms (RAPD) using a modified polymerase chain reaction (PCR, Sa...