Periwinkle (Catharanthus roseus), a member of the Apocynaceae family, is highly valued in the horticultural industry. A native to Madagascar, this herbaceous plant grows to ≈80 cm high and blooms continuously year-round with pink, purple, or white flowers (Hogan, 2003). Study of periwinkle has increased because of its ability to produce secondary metabolites such as terpenoid indole alkaloids that may be used to treat cardiac diseases and certain tumors in mammals (Favali et al., 2004). Periwinkle is also a natural host of many phytoplasmas. Phytoplasmas are cell wall-less organisms that infect over 300 plant species, including many woody species. They thrive in the phloem of infected plants and can cause symptoms such as curled and yellow leaves, witches-broom appearance, and phyllody, resulting in reduced crop yield and plant death (Lee et al., 2000). One phytoplasma disease, called X-disease, causes severe damage on many Prunus species such as sweet and sour cherries, peach, nectarine, and chokecherry (Guo et al., 1996). Periwinkle is a small, relatively fast-growing plant, making it an ideal species for the study of pathogen–host interaction of phytoplasma. Development of an efficient transformation system used for testing genes is important to elucidate the function of future-cloned resistant genes and interaction between phytoplasma and plant hosts.
A regeneration system is a prerequisite for recovering transgenic plants. Many factors, including genotype (Choi et al., 2004; Obukosia et al., 2005; Xu et al., 2007), medium (Trigiano and Gray, 2000), explant type (Gambino et al., 2007), plant growth regulator (PGR) (Trigiano and Gray, 2000), and culture condition (Choi et al., 2001), can affect plant regeneration.
Periwinkle organogenesis was first reported in the late 1970s by Dhruva et al. (1977) followed by Ramavat et al. (1978) and Abou-Mandour et al. (1979). However, the shoot regeneration rate was low. In 1989, Mollers and Sarkar induced calluses from phytoplasma-infected stem tissues. These callus tissues differentiated into plants on the MS medium with 0.25 mg·L−1 6-benzyladeine (BA), 1.0 mg·L−1 1-naphthalene acetic acid (NAA), and 10.0 mg·L−1 gentamicin. Further test confirmed that phytoplasmas were eliminated. Recently, efficient plant generation of periwinkle was accomplished mainly through somatic embryogenesis starting with generative tissues such as anthers and immature zygotic embryos. Kim et al. (1994) induced somatic embryos from calluses derived from anthers in MS medium supplemented with 1.0 mg·L−1 NAA and 0.1 mg·L−1 kinetin. Plants were also regenerated from immature zygotic embryos in 2, 4-D containing MS medium through a callusing phase (Kim et al., 2004). An efficient somatic embryogenesis system has been established. Embryogenic calluses were developed from hypocotyls and primary cotyledonary somatic embryos and somatic embryos were then differentiated on medium supplemented with 1.0 mg·L−1 NAA and 1.5 mg·L−1 BA (Junaid et al., 2007). However, the relatively high regeneration rate published was achieved through somatic embryogenesis and only one cultivar was used.
The objective of this research was to determine factors such as PGR, genotype, explant, dark treatment, and antibiotics that influence plant regeneration ability of periwinkle. Such data are essential for the successful development of an efficient plant regeneration system for Agrobacterium-mediated transformation in this species.
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