Agrobacterium-mediated genetic transformation of radish (Raphanus sativus L.)

Abstract

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...

Advances and Milestones of Radish Breeding: An Update

“…The use of adventitious shoot growth on hypocotyl explants for Agrobacteriummediated radish genetic transformation was investigated using transgenic radish (Raphanus sativa L., cv. Jin Ju Dae Pyong) grown on Murashige and Skoog medium (Cho et al 2008). Besides, northern blot results revealed the GUS gene transcript was detected in few regenerated plants hence, confirming genetic transformation (Cho et al 2008).…”

“…Jin Ju Dae Pyong) grown on Murashige and Skoog medium (Cho et al 2008). Besides, northern blot results revealed the GUS gene transcript was detected in few regenerated plants hence, confirming genetic transformation (Cho et al 2008).…”

Efficient <i>Agrobacterium</i>-mediated genetic transformation method using hypocotyl explants of radish (<i>Raphanus sativus</i> L.)

“…of transgenic shoots per hypocotyl explants) of GV3101 was higher than that of the EHA101, and the mean value (GV3101 : 13.3% and EHA101 : 6.2%) also supported the difference. Thus, we could improve the transformation efficiency from 0.26% that was previously described in the publication (Cho et al 2008) to 13.3% using the "Pirabikku" hypocotyl as the explant source. Four transgenic shoots transformed by Agrobacterium strain GV3101 were transferred to shoot elongation medium (B5 medium containing 1 mg l −1 gibberellic acid, 10 mg l −1 hygromycin, 200 mg l −1 carbenicillin, 10 g l −1 sucrose, and 4 g l −1 gellan gum with pH 5.8), and these plants were further grown in a root induction medium (B5 medium containing 2 mg l −1 indole-3-butyric acid, 10 mg l −1 hygromycin, 200 mg l −1 carbenicillin, 10 g l −1 sucrose, and 4 g l −1 gellan gum with pH 5.8).…”

“…Although these studies have accelerated the investigation of the correlation between the genes and traits, molecular breeding based on the revealed molecular function of these genes has not been developed because a genetic transformation system with high efficiency and reproducibility has not been constructed in Raphanus. In some previous publications on Raphanus transformation, many different methods such as the floral dip (Curtis and Nam 2001), ultrasonic and vacuum infiltration (Park et al 2005), and Agrobacterium-mediated genetic transformation using the hypocotyl explants (Cho et al 2008) have been reported. However, in these methods, the transformation efficiency is insufficient because the number of transgenic plants produced in one experiment is relatively small, which makes the molecular breeding of radish with practical traits unrealistic.…”

“…The hygromycinresistant shoots regenerated were transferred to a shoot maturation medium (B5 medium composed of 10 mg l −1 hygromycin, 200 mg l −1 carbenicillin, and 10 g l −1 sucrose, 4 g l −1 gellan gum with pH 5.8) for 6 weeks (Figure 1B). We compared the transformation efficiency of Agrobacterium strains EHA101 and GV3101 because a recent study reported that GV3101 was more efficient in transforming radish (Cho et al 2008). Hypocotyl explants were infected with an Agrobacterium strain GV3101 harboring pIG121-Hm in the same way as EHA101.…”