Highly regenerative callus was isolated from the base of adventitious shoots on cotyledon explants of Dianthus hybrida Telstar Scarlet cultured on MS medium supplemented with 1 mg l )1 TDZ and 0.1 mg l )1 NAA. Flow cytometric analysis showed that cotyledon tissue is a mixture of diploid and tetraploid cells. Whereas the regenerative callus consisted of cells showing various ploidy levels of 2C to 16C, their regeneration ability was maintained as long as they were sub-cultured onto fresh media. More than 93% of regenerated shoots from the calluses were diploid. Only a few shoots were revealed as tetraploids and octoploids, suggesting that diploid cells had higher regeneration ability.Abbreviations: BA -benzyladenine; MS -Murashige & Skoog; NAA -naphthalenacetic acid; TDZthidiazuron Regenerative callus culture is suitable for massproduction of plants in a limited space. However one restriction to callus culture, the most controversial point, is the genetic stability of plants regenerated from callus: they often present somaclonal variations. These somaclonal variations are attributable to cytogenetical changes including polyploidism and aneuploidism.Dianthus hybrida Telstar Scarlet is an interspecific hybrid between D. chinensis and D. barbatus that is used for potted plants and bedding plants. Shoot regeneration from callus-derived protoplasts of Telstar Scarlet has been reported (Nakano and Mii, 1993), suggesting a high regeneration ability of this genotype. No reports have addressed ploidy variations of callus cultures in genus Dianthus whereas successful shoot regeneration has been reported (Kallak et al., 1997). Efficient and reliable regeneration system will allow us to study on genetic transformation in Dianthus species. This paper describes regenerative callus of Dianthus Telstar Scarlet that were isolated from cotyledons, as well as ploidy level variations in calluses and regenerated shoots.Seeds of Telstar Scarlet were sown aseptically on filter paper, with sterile water in vitro, then incubated for 5 days in the dark at 25°C. Distal ends of cotyledons of germinated seedlings were cut into 3 mm 2 explants. The explants were cultured on MS medium (Murashige and Skoog, 1962) supplemented with various combinations of cytokinins (BA, TDZ, or Zeatin; 1.0 or 5.0 mg l )1 ) and auxins (IAA or NAA; 0.01 or 0.1 mg l )1 ). We cultured 20 explants on 25 ml medium in petri dishes (15 · 90 mm); five replications were made per treatment. All cultures were incubated at 25°C under a 16-h photoperiod at 36 lmol m )2 s )1 provided by cool white fluorescent lamps for 4 weeks.Cotyledon explants cultured on all media except 5 mg l )1 BA produced calluses. Only a small fraction of explants produced adventitious

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Carnation is a valuable crop for the cut flower industry and demand for new and improved varieties is growing. However, genetic transformation of carnations is currently limited because of a lack of efficient routine technique. In this chapter, we present an easy and effective protocol for gene transfer to carnation node explants and subsequent adventitious shoot regeneration. For high-adventitious shoot regeneration, node explants from first to third node of 5- to 8-cm long shoots were cultured on Murashige and Skoog (MS) medium, containing 1.0 mg/Lthidiazuron (TDZ), 0.1 mg/L alpha-napthalenoacetic acid (NAA), 20 g/L sucrose, and 2 g/L Gellan gum for 10 d. Then the explants were cut into 8 radial segments and subcultured onto MS medium, containing 1.0 mg/L BA, 0.1 mg/L NAA, 20 g/L sucrose and 2 g/L Gellan Gum. For effective genetic transformation, 3- to 5-d precultured node explants were submerged in an Agrobacerium suspension for 10 min, then cocultivated on filter paper soaked with water and 50 microM acetosyringone (AS). After cocultivation, the explants were cut into eight radial segments and subcultured onto selection medium until transformed shoots regenerated from the explants.

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This chapter describes a rapid and efficient protocol for explant preparation and genetic transformation of carnation. Node explants from greenhouse-grown plants and leaf explants from in vitro plants are infected with Agrobacterium tumefaciens AGL0 harboring pKT3 plasmid, consisting of GUS and NPTII genes. Explant preparation is an important factor to obtain the transformed plants. The GUS-staining area was located only on the cut end of explants and only explants with a cut end close to the connecting area between node and leaf, produced transformed shoots. The cocultivation medium is also an important factor for the successful genetic transformation of carnation node and leaf explants. High genetic transformation efficiency of node and leaf explants cocultured with Agrobacterium tumefaciens was achieved when the explants were cocultivated on a filter paper soaked with water or water and acetosyringone mixture (AS).

Bacterial wilt disease caused by the soil-borne bacterium, Ralstonia solanacearum species complex (formerly Pseudomonas solanacearum), causes great yield losses (Elphinstone, 2005). Because of the broad host range of and the limited control measures for R. solanacearum species complex, which attacks more 200 species representing 50 families of plants including the Solanaceae, Musaceae, Zingiberaceae and Cucurbitaceae (Hayward, 1994;She et al., 2017), the disease is difficult to manage. Bacterial wilt is a common disease in tropical, subtropical and warm temperate regions of the world. The disease is known to cause substantial damage to the economic plant production . The initial symptom is a drooping of leaves.Affected leaves later become permanently wilted, as the disease develops, a brown discoloration of xylem vessels in the stem is observed. A creamy or slimy mass of bacteria exudes from cut or broken vascular bundles. Eventually, plants fail to recover and become yellow and then necrotic (Charkowski et al., 2020;Dhital et al., 2000).In traditional classification schemes, R. solanacearum was currently classified into four biovars based on the oxidization of three