Eggplants transformed with Sw-5 gene, regenerated by organogenesis and somatic embryogenesis, were resistant to the Tomato chlorotic spot virus, while wild plants did present systemic infection. TO plants were selfed and the segregation analysis of T1 and T2 generation indicated the existence of one or more insertion sites. Southern blot analysis ...confirmed one or two independent insertions in T2 plants. Different lesions associated with the insertion number were observed in TI and T2 plants. T2 plants with two copies displayed faster hypersensitive reactions and characteristic necrotic lesions that contrasted with slower responses and necrotic ring lesions in plants with one copy. These results suggest that the Sw-5 confers resistance to tospovirus in transgenic eggplants and that the resistant phenotype depends on the number of transgene copies.
The effects of kanamycin and hygromycin-based selection and co-culture temperature ranging from 22 to 28 °C upon eggplant transformation efficiency were evaluated. Both morphogenic pathways, somatic embryogenesis and organogenesis, were adopted using cotiledonary and hypocotyl explants, respectively. Somatic embryos were recovered in the presence... of both antibiotics, although lesser escapes were observed in hygromycin-supplemented medium. Indeed, selection provided by this antibiotic was more efficient compared to kanamycin, nevertheless, shoot regeneration was not observed with hygromycin. Significant difference on the frequency of cotiledonary explants displaying callus (FEC) was observed as embryogenesis was concerned, although a higher number of embryos was observed in hygromycin selective media. The frequency of explants presenting callus (FEC), embryos (FEE) and shoots or buds (FERG) did not differ statistically for the tested co-culture temperatures, although higher regenerant number was observed at 24 °C.
Cotyledon protoplasts were isolated from 16-18-day-old in vitro grown seedlings of 9 Brazilian and 3 Hungarian pepper varieties and hybrids. Large numbers (average 9.59 X 106 protoplasts g 14 fresh weight) of highly viable (average 87.0%) protoplasts were released using a pectocellulolytic enzyme mixture. Protoplasts were cultured in K8p mediuni u...sing an alginate disc embedding method. The osmotic pressure of the medium surrounding the alginate-embedded protoplasts was reduced by replenishing the liquid medium at K8p:K8 ratios of 1:0. 2:1, 1:1 in the first. second, and third week, respectively. Initial plating efficiency (IPE) average was 38.5% and after 21 days protoplasts reached microcolonies (15-20 cells) stages. Microcolonies were transferred after 3-4 weeks to a MS-based medium supplemented with 1.0 mg I-1 zeatin, 3.0% (w/v) sucrose, 0.24% (w/v) phytagel and pH 5.8, whereupon they formed callus. Final plating efficiency (FPE) average was 0.29% at a plating density of 1.0 x 105 protoplasts Protoplast-derived calli were cultured on a range of MS-based media supplemented with either BAP, IAA, TDZ; and zeatin. No morphogenic response was observed in any genotype investigated.