Efficient floral dip transformation method using Agrobacterium tumefaciens on Cosmos sulphureus Cav.

Yellow cosmos (Cosmos sulphureus Cav.) is a specific flowering plant and considered a suitable genetic engineering model. Agrobacterium-mediated plant transformation is commonly used for plant genetic engineering. Floral dip transformation is one of the plant genetic transformation methods, and it involves dipping flower buds into an Agrobacterium suspension. Studies on floral dip transformation of yellow cosmos have never been reported. Therefore, an efficient method in plant genetic engineering must be established. This study developed an effective and efficient floral dip transformation method for yellow cosmos. In this study, flower buds with sizes of 5-7 mm were used. Several parameters have been observed to optimize the floral dip method. These parameters included the optical density (OD600) of Agrobacterium culture, concentration of surfactant, and duration of flower bud dipping into the Agrobacterium suspension. The results showed that the floral dip method was most efficient when the flower buds were dipped into Agrobacterium suspension with OD600 = 0.8 and containing 5% sucrose and 0.1% Silwet L-77 for 30 s. This method enhanced the transformation efficiency at a rate of 12.78 ± 1.53%. The neomycin phosphotransferase II and green fluorescent protein genes with sizes of 550 and 736 bp, respectively, were confirmed by polymerase chain reaction. In addition, the transgenic plants were kanamycin resistant and fluorescent under ultraviolet light observation. This finding suggests that the proposed floral dip transformation provides new insights into efficient plant genetic engineering methods for yellow cosmos.

The Divergence of Chromosome Structures and 45S Ribosomal DNA Organization in Cucumis debilis Inferred by Comparative Molecular Cytogenetic Mapping.

Cucumis debilis W.J.de Wilde & Duyfjes is an annual and monoecious plant. This species is endemic to Southeast Asia, particularly Vietnam. However, C. debilis is rarely studied, and no detailed information is available regarding its basic chromosome number, 45S ribosomal DNA (rDNA) status, and divergence among other Cucumis species. In this study, we characterized the morphological characters and determined and investigated the basic chromosome number and chromosomal distribution of 45S rDNA of C. debilis using the fluorescent in situ hybridization (FISH) technique. A maximum likelihood tree was constructed by combining the chloroplast and internal transcribed spacer of 45S rDNAs to infer its relationship within Cucumis. C. debilis had an oval fruit shape, green fruit peel, and protrusion-like white spots during the immature fruit stage. FISH analysis using 45S rDNA probe showed three pairs of 45S rDNA loci located at the terminal region in C. debilis, similar to C. hystrix. Meanwhile, two, two, and five pairs of 45S rDNA loci were observed for C. melo, C. metuliferus, and C. sativus, respectively. One melon (P90) and cucumber accessions exhibited different chromosomal localizations compared with other members of Cucumis. The majority of Cucumis species showed the terminal location of 45S rDNA, but melon P90 and cucumber exhibited terminal-interstitial and all interstitial orientations of 45S rDNA loci. Based on molecular cytogenetics and phylogenetic evidence, C. debilis is more closely related to cucumber than melon. Therefore, C. debilis may serve as a potential parental accession for genetic improvement of cucumber through interspecific hybridization.

Inheritance and Realized Heritability of Resistance to Imidacloprid in the Brown Planthopper, Nilaparvata lugens (Hemiptera: Delphacidae), From Indonesia.

The brown planthopper, Nilaparvata lugens Stål., is a major insect pest causing serious damage to the rice crop in many rice-producing countries. The aim of the study was to determine the inheritance characteristics and realized heritability of resistance to imidacloprid in N. lugens from Banyumas, Central Java Province, Indonesia. The five generations of selection increased the resistance ratio (RR) from 46.20-fold at generation 45 (G45) to 150.39-fold (G49) compared with the laboratory-susceptible population. The LC50 values of imidacloprid for the progeny of reciprocal crosses, F1 and F1', showed no significant differences, indicating that imidacloprid resistance was inherited autosomally in N. lugens. The degree of dominance (D) values for F1 and F1' were 0.65 and 0.64, respectively, suggesting that imidacloprid resistance in N. lugens was expressed as an incompletely dominant trait. Chi-square analysis based on the monogenic hypothesis indicated that imidacloprid resistance in N. lugens was polygenic. Furthermore, the estimation realized heritability value (h2) was 0.0893. These results would be useful to determine an effective resistance management strategies in N. lugens.