Radish (Raphanus sativus L.) belongs to the family Brassicaceae, and is an important plant that is cultivated throughout Asia. Korean varieties are rich in vitamins B and C and have important medicinal properties. However, many of these varieties are susceptible to various pathogens and conventional breeding is not successful in introducing resistance genes into the varieties because of limited availability of germplasm. Alternatively, it is required to establish a genetic transformation system of this species to transfer resistance genes from various sources rather than radish germplasm. The importance and needs for the genetic improvement of radish have recently been reviewed by Curtis (2003). A floral-dip method previously developed for genetic transformation of Arabidopsis (Clough and Bent 1998) was used for radish, resulting in genetic transformation frequencies of 1.1%-1.4% (Curtis and Nam 2001;Curtis et al. 2002). However, transgenic plants produced only 50-100 seeds per individual. Furthermore, many of the plants were siblings (Curtis et al. 2004). Therefore, Agrobacterium-mediated transformation might be more appropriate to this species as demonstrated in a vast array of other crops. A few efficient adventitious shoot regeneration systems were developed for Japanese (Matsubara and Hegazi 1990), Chinese (Pua et al. 1996), and Korean ecotypes of radish (Curtis et al. 2004). However, there have been no reports on production of transgenic radish by Agrobacteriummediated transformation. This paper describes Agrobacterium-mediated genetic transformation of radish using adventitious shoot formation on hypocotyl explants.Zygotic embryos of F1 hybrid radish (Raphanus sativus L. cv. Jin Ju Dae Pyong) were used. Seeds were surface disinfected with 70% ethanol for 1 min and then with 0.4% sodium hypochlorite solution for 15 min. They were rinsed three times with sterile deionizeddistilled water before being placed on MS medium (Murashige and Skoog 1962) without growth regulators contained in Petri dishes. The pH of all media was adjusted before autoclaving. Twenty-five ml of medium was dispensed into 90ϫ15-mm plastic Petri dishes. After 5 to 7 days of incubation in the dark, hypocotyls, 4 to 6-cm long, were decapitated from 5 to 7 day old seedlings and then cut into 5 to 7-mm long segments (hypocotyl explants). To determine the optimum concentration and combination of growth regulators for adventitious shoot formation, hypocotyl explants were cultured on MS Abstract In order to generate transgenic radish (Raphanus sativus L., cv. Jin Ju Dae Pyong), hypocotyl explants were cultured on Murashige and Skoog medium containing 4 mg l Ϫ1 AgNO 3 , 5 mg l Ϫ1 acetosyringone, 4 mg l Ϫ1 6-benzyladenine, and 3 mg l Ϫ1 a-naphthaleneacetic acid in addition to either 10 mg l Ϫ1 hygromycin or 100 mg l Ϫ1 paromomycin after cocultivation with disarmed Agrobacterium tumefaciens harboring a plant expression binary vector. Explants co-cultivated with A. tumefaciens GV3101 harboring pCAMBIA1301 and A. tumefaciens EHA101 harboring pPTN290...
A DNA extraction method using Chelex 100 is widely used for bacteria, Chlamydomonas, and animal cell lines, but only rarely for plant materials due to the need for additional time-consuming and tedious steps. We have modified the Chelex 100 protocol and successfully developed a rapid and simple method of DNA extraction for efficient PCR-based detection of transgenes from a variety of transgenic plant and algal species. Our protocol consists of homogenizing plant tissue with a pestle, boiling the homogenized tissue in a microfuge tube with 5% Chelex 100 for 5 min, and centrifuging the boiled mixture. The supernatant, which is used for PCR analysis, was able to successfully amplify transgenes in transgenic tobacco, tomato, potato, Arabidopsis, rice, strawberry, Spirodela polyrhiza, Chlamydomonas, and Porphyra tenera. The entire DNA extraction procedure requires \15 min and is therefore comparable to that used for bacteria, Chlamydomonas, and animal cell lines.
An efficient in vitro protocol has been established for somatic embryogenesis and plantlet conversion of Korean wild ginseng (Panax ginseng Meyer). Wild-type and mutant adventitious roots derived from the ginseng produced calluses on Murashige and Skoog (MS) medium supplemented with 0.5 mg/L 2,4-dichlorophenoxyacetic acid and 0.3 mg/L kinetin; 53.3% of the explants formed callus. Embryogenic callus proliferation and somatic embryo induction occurred on MS medium containing 0.5 mg/L 2,4-dichlorophenoxyacetic acid. The induced somatic embryos further developed to maturity on MS medium with 5 mg/L gibberellic acid, and 85% of them germinated. The germinated embryos were developed to shoots and elongated on MS medium with 5 mg/L gibberellic acid. The shoots developed into plants with well-developed taproots on one-third strength Schenk and Hildebrandt basal medium supplemented with 0.25 mg/L 1-naphthaleneacetic acid. When the plants were transferred to soil, about 30% of the regenerated plants developed into normal plants.
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