Tachyplesin I is a 2.3 kDa antimicrobial peptide isolated from Southeast Asian horseshoe crabs. Bacterial suspensions containing 1 x lo6 colony-forming units/ml of six isolates of pectolytic Erwinia spp., the causal pathogens of potato soft rot and blackleg, were killed in vitro by 1.4 to 11.1 fig/ml of tachyplesin I. In an attempt to enhance resistance to Erwinia spp., each of the potato cultivars Bintje, Karnico and Kondor were transformed with two gene constructs encoding different precursor tachyplesin I proteins under the control of a cauliflower mosaic virus 35s promotor. Northern and western blot analysis showed that the tachyplesin I gene was expressed in transgenic plants. Small tubers of 17 transgenic clones were screened twice for soft rot resistance to Erwinia carotovora ssp. atraseptica. Under aerobic or anaerobic conditions, transgenic clones showed slightly less rot than control tubers.

Potato‐tuber slices and small tubers of 12 cultivars were inoculated with isolates of Erwinia carotovora subsp. atroseptica (Eca), E. c. subsp. carotovora (Ecc) and E. chrysanthemi (Ech). Cultivar resistance was expressed as the mean diameter of the rotted‐tissue area. Grouping of the cultivars into classes of relatively high, intermediate or low resistance, was different after incubation in air in comparison to incubation under anaerobic conditions, and depended on the (sub‐)species of bacteria used. When compared to the effect of inoculation with different (sub‐)species, grouping of the cultivars was less affected by isolates within (sub‐)species. Cultivar grouping was hardly affected by site of inoculation, being either in the medullary tissue or in the cortex. In general, the results of experiments with either field‐grown seed tubers or glasshouse‐grown small tubers from 2 years were reproducible, except when tuber tissue was inoculated with Ech and incubated, aerobically, in which case cultivars only showed relatively small differences for resistance. Correlation coefficients between the results from slice and small‐tuber screenings were 0.71 and 0.64 for Eca and Ecc, respectively. This implies, some potential for applying the method in the early stages of potato‐breeding programmes.The results of this study show that soft‐rot resistance of clones to different pectolytic Erwinia spp. can be determined efficiently and reproducibly if the oxygen concentration during incubation is standardized.

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In t991 and 1992, 12 potato cultivars were screened at two locations for resistance to blackleg, after vacuum infiltration of the seed with Erwinia carotovora subsp, atroseptica or E. chrysanthemi. Cultivar differences for resistance to E.c. subsp, atroseptica and E. chrysanthemi were found which were consistent over locations and years. Seed tubers of the same cultivars were also screened for resistance to both Erwinia spp. by using a tuber slice inoculation method. Correlation coefficients for comparisons between resistance to blackleg in the field and tuber tissue resistance under aerobic or anaerobic conditions were not significant. This could partly be explained by drastic changes in relative tuber tissue resistance of the cultivars within a 5 weeks period after planting in the field. Presprouting of seed tubers in diffuse daylight had a less pronounced effect on relative tuber tissue resistance than planting in the field. Monitoring the process of mother tuber decay during the growing season of 1993 after vacuum infiltration with E.c. subsp, atroseptica and E. chrysanthemi revealed that cultivars differed in the extent to which these bacteria enhanced the process of mother tuber decay. These differences partly explained the cultivar differences for resistance to blackleg in the field.Abbreviations: Eca = Erwinia carotovora subsp, atroseptica; Ech = Erwinia chrysanthemi; NOP = Noordoostpolder; Wag --Wageningen

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4 x 10(6) cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.