Plant Cell Reports (1995) 14:648-651

PlantCell Reports
9 Springer-Verlag 1995

Somatic embryogenesis and plant regeneration in tissue cultures of radish (Raphanus sativus L.)
Won J. Jeong, Sung R. Min, and Jang R. Liu
BioresourcesResearchGroup, Genetic EngineeringResearch Institute, K1ST, P.O. Box 115, Taedok ScienceTown, Taejon, 305-600 Korea Received4 October 1994/Revised version received4 January 1995 - Communicatedby E Constabel

Abstract. Hypocotyl segments of 2to 3-week-old radish (Raphanus sativus L. cv. Fa H a n d s o m e Fall) seedlings produced yellowish compact calli w h e n cultured on Murashige and Skoog's (MS) m e d i u m supplemented with 1 mgl "1 2,4-dichlorophenoxyacetic acid (2,4-D). Upon transfer onto medium containing 6-benzyladenine and a-naphthaleneacetic acid, up to 5.3% of the calli gave rise to a few somatic embryos. W h e n subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced w h i t e , compact calli which formed numerous embryos. These calli maintained their embryogenic capacity for over 18 months. When cultured on m e d i u m containing 0.1 to 3 mgl 1 2,4-D, up to 90% of longitudinally sliced somatic embryo halves produced calli with numerous secondary embryos. Embryos were transferred onto m e d i u m containing 0.1 mgl "1 2,4-D and 1 mgl "1 abscisic acid where they developed into the cotyledonary stage. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets. These plantlets were successfully transplanted in potting soil and after cold treatment they were grown to maturity in a phytotron. Key words: Embryogenic callus - culture - - Somatic embryos
Plant tissue

Many members of the Cruciferae are amenable to regeneration from cultured ceils and tissues via organogenesis. Plant regeneration via somatic embryogenesis in tissue cultures other than from anthers and microspores has been only rarely described in this family (Zee and Johnson 1985). Such is the case for radish (Raphanus sativus L.), one of the most important vegetables in China, Japan, and Korea where plant regeneration via organogenesis from seedling explants and somatic embryogenesis from anthers and microspores have been achieved (Matsubara and Hegazi 1990; Lichter 1989). However, the frequency of plant regeneration in these systems is too low to be of practical use for maintenance and proliferation of self-incompatible parental lines, which are indispensible for F1 hybrid seed production. A reliable plant regeneration system is also required for future incorporation of agronornically useful foreign genes by genetic transformation. This study describes culture conditions for somatic embryogenesis and plant regeneration from hypocotyl explants in radish.

Materials and methods

Seeds of radish (R. sativus L. cv. F1 Handsome Fall) were surface-sterilized with 70% ethanol for 5 rain and a 1% sodium hypochlorite solution for 30 rain. They were then rinsed with sterilized double-distilled water 3 times, and incubated on Murashige and Skoog's (MS) (1962) basal medium at 25"(3 in the dark. Unless mentioned otherwise, 10 explants (or seeds) were placed in a 87 x 15-mm plastic Petri dish containing 25 ml of medium solidified with 0.6% Phytagel. After 2 to 3 weeks of incubation, hypocotyls of the germinated seedlings were sliced into 5-ram-long segments and placed on MS medium supplemented with 1 mgla 2,4-dichlorophenoxyacetic acid

Abbreviation: 2,4-D, 2,4-dichlorophenoxyacetic acid; BA, 6-benzyladenine; GA3, gibberellin A3; IAA, indole-3-acetic acid; MS, Murashige and Skoog; NAA, a-naphthaleneacetic acid Introduction
Correspondence to: J.R. Liu

649
(2,4-D). All cultures were maintained at 25"C in the light (a 16-h photoperiod; approximately 5 Win2; cool-white fluorescent tubes). After 5 weeks of culture, calli formed on the cut surfaces of hypocotyl segments. The calli were isolated and transferred to MS medium with a factorial combination of 6-benzyladenine (BA) (0, 0.1, 0.3, 1 or 3 mgl a) and a-naphthaleneacetic acid (NAA) (0, 0.1 or 0.3 mgl'l), and cultured to induce somatic embryos. Each treatment involved 5 Petri dishes with 10 calli (3 mm in diameter) per dish. The isolated initial calli were also maintained by subculturing on medium containing 1 mgla 2,4-D every four weeks. The frequency of somatic embryo formation or callus formation was determined after 3 weeks of culture by counting the number of explants forming somatic embryos or calli per dish. Somatic embryos were graded by measuring their size with a ruler as follows: K2 mm, 2.1-3 mm, 3.1- 4 mm, and >4 m m long. To produce secondary embryos, somatic embryos were sliced longitudinally into two halves. Ten halves per dish, 40 in total, were cultured on medium containing 0.1, 0.3, 1 or 3 mgla 2,4-D under the same conditions as above. After 2 weeks of culture, caUi (3 m m in diameter) which formed on the somatic embryo halves were isolated and transferred to medium containing 0, 0.1, 0.3, 1 or 3 mgla 2,4-D, and cultured to assess their embryogenic capacity. The frequency of secondary embryo formation or embryogenic callus formation was determined as above. For histological studies, somatic embryos were fixed in 2% glutaraldehyde for 5 h, dehydrated in a tertiary-butanol series, and embedded in paraffin. Serial sections were cut (7 /~m in thickness) and stained with 0.5% hematoxylin (Sass, 1971). To regenerate plantlets, somatic embryos were transferred onto MS basal medium, medium containing 1 mgla gibberellin A3 (GAa) or medium containing 0.1 mgl a 2,&D and 1 mgl "~ abscisic acid (ABA) and cultured in the light. Plantlets were transplanted in potting soil and grown in a phytotron (a 16-h photoperiod; approximately 100 Wm -2 23"G day/20~ night) for 2 to 8 weeks. The regenerants were bolted by lowering the temperature to 8 "C for 4 weeks of a 12-h photoperiod, and resumed growth by restoring the previous environmental conditions.

when cultured on the various media described above, indicating that they are nonmorphogenic. However, when subcultured for 3 to 6 months, 7% of the yellowish, compact calli produced minor, white, compact calli forming numerous embryos. When subcultured every four weeks, these white compact calli maintained their embryogenic capacity for over 18 months. Approximately 90% of the somatic embryos derived from these calli had one trumpet-shaped aberrant cotyledon, and the others typically had
t w o cotyledons. After 2 w e e k s of culture, o v e r 50% of the somatic embryo halves began to produce n u m e r o u s s e c o n d a r y s o m a t i c e m b r y o s at the cut

Results
After 2 weeks of culture, over 95% of the hypocotyl segments formed yellowish, compact calli on their cut edges (Fig. 1A). After 5 weeks of culture, the size of calli increased two to three-fold. The hypocotyl segments also produced minor, off-white, friable calli. Upon transfer to
m e d i u m containing BA a n d N A A , the yellowish, c o m p a c t calli sporadically g a v e rise to a f e w somatic e m b r y o s (Fig. 1B) at frequencies of u p to 5.3%. T h e s e calli d i d not p r o d u c e somatic e m b r y o s w h e n transferred to the basal m e d i u m (Fig. 2). The friable calli f r o m h y p o c o t y l segments w e r e n o t c a p a b l e of f o r m i n g organized structures

Fig. 1. Somatic embryogenesis and plant regeneration of radish (cv. Handsome Fall F1). A: Yellowish, compact calli formed on hypocotyl segment cut edtges cultured on MS medium supplemented with 1 mgl" 2,4-D; B: Somatic embryos derived from an embryogenic callus on MS medium with BA and NAA; C: Numerous secondary embryos arising directly from an initial somatic embryo half cultured on MS medium with 0.1 mgl"~ 2,4-D; D: Longitudinal section of a torpedo-shaped somatic embryo; E: Plantlets regenerated from somatic embryos cultured on MS basal medium; F: A potted regenerant grown in a phytotron; G: A flowering regenerant. Scale bar = 1 ram.

650 edges and the intact surfaces formed somatic embryos directly or, in a few cases, with an intervening callus (Fig. 1C). A few embryo halves formed translucent, friable calli which were not capable of producing embryos. After 3 weeks of culture, the frequency of direct secondary embryo formation on somatic embryos halves shorter than 4-ram-long was 5 to 30% higher than on longer halves (more advanced) in various concentrations (0.1 to 3 mgl "1) of 2,4-D.
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embryos when transferred onto medium containing up to 0.3 mgl "1 2,4-D (data not shown). Histological examination of torpedo-shaped embryos revealed a bipolar organization with an integrated shoot-root axis (Fig. 1D). Upon transfer to MS basal medium or medium containing 1 mgl "1 GA3, somatic embryos from white, compact calli developed into planflets at a frequency of approximately 20% after 2 weeks of culture. (The frequency was not noticeably increased by addition of GA3.) To enhance the conversion frequency, somatic embryos derived from white, compact calli were transferred onto medium containing 0.1 mgl "1 2,4-D and 1 mgl"1 ABA where they developed into the cotyledonary stage after 3 weeks of culture. Upon transfer onto half-strength MS basal medium, approximately 90% of the embryos developed into plantlets (Fig. 1E). Hantlets were successfully transplanted in potting soil and grown in a phytotron (Fig. 1F).
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Fig. 2. Frequency of somatic embryo formation on radish (cv. Handsome Fall F1) hypocotyl-derived yellowish, compact calli. Ten caUi (3 mm in diameter) with 5 replicates per treatment were cultured and data were collected after 3 weeks of culture. Bars indicate standard errors.

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The frequency decreased as the concentration of 2,4-D was increased (Fig. 3A). However, the frequency of embryogenic callus formation on somatic embryos halves increased as the developmental stage advanced up to 3 to 4 mm in length, and as the concentration of 2,4-D in the medium was increased up to 1 mgl "1 (Fig. 3B). Secondary embryos were capable of undergoing development into the torpedo stage on medium containing 3 mgl "1 2,4-D, indicating that their development was not significantly inhibited by the 2,4-D concentration. Approximately 90% of mature secondary embryos also had one ~umpet-shaped cotyledon, and developed into plantlets under the same conditions as above. Embryogenic calli which formed on somatic embryo halves were subcultured on medium containing 1 mgl "1 2,4-D every 4 weeks, and they maintained their embryogenic capacity for over 18 months. Subcultured calli gave rise to numerous somatic

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Fig. 3. Frequency of secondary embryo formation (A) and embryogenic callus formation 03) on somatic embryos derived from radish (cv. Handsome Fall Fz) hypocotyl segments when cultured on medium containing various concentrations of 2,4-D. Somatic embryos were longitudinally sliced to halves before culture. Ten halves with 4 replicates per treatment were cultured and data were collected after 3 weeks of culture. Bars indicate standard errors.

651 After the cold treatment, all of the regenerants were bolted. Twenty cold-treated regenerants flowered 1 to 2 months after restoring growth and were morphologically normal (Fig. 1G). culture is most practical for this purpose, plant regeneration via organogenesis from seedling explant-derived calli has been achieved (Matsubara and Hegazi, 1990). However, the frequency of plant regeneration is too low to be practical. The culture conditions for somatic embryogenesis and plant regeneration described in this study may provide a routine way to maintain and proliferate self-incompatible lines at a lower cost than shoot-tip culture. Also, in order to introduce agronomicaUy useful foreign traits, such as herbicide, insect, and disease resistance into this species using recently advanced genetic transformation techniques, the experimental protocol described in this study may be applicable. A high frequency of transgenic secondary somatic embryo formation may be expected by co-culturing initial somatic embryo segments with Agrobacterium, as demonstrated with cassava (Chavarriaga-Aguirre et al. 1992).

Discussion
In general, embryogenic callus does not require growth regulators in a culture medium to develop into somatic embryos (Ammirato, 1984). However, in this study either BA or NAA was indispensible for the development of somatic embryos from initial yellowish, compact calli in radish. This result is in agreement with results from studies with rice (Jeong et al., 1991). However, white, compact calli from the initial calli and somatic embryo halves were capable of producing numerous embryos when maintained on m e d i u m containing 2,4-D. The result is more typical of other species such as cassava (Szabados et al., 1987) and Codonopsis (Min et al., 1992). We suggest that the embryogenic capacity of the initial callus is unstable, but it is then stabilized by repeated subculture. This stabilization of an initial embryogenic callus by repeated subculture has also been reported in wheat (Vasil et al., 1990). However, the possibility that some embryogenic ceils were embedded in initial callus and proliferated to emerge with time in culture cannot be excluded. In addition, the development of secondary embryos was not significantly inhibited by 2,4-D, indicating that they were less sensitive to 2,4-D than the initial somatic embryos. Cotyledonary explants were also cultured under the same conditions as above. However, the frequency of embryogenic callus formation was lower than 1%. Preculture of somatic embryos on medium containing ABA promoted conversion of embryos into plantlets. This was probably due to an enhanced maturity of embryos by ABA. Seedling explants were cultured from several other cultivars, including All Season Altari (F1 hybrid). However, none of the cultivars produced somatic embryos, suggesting that the competence of somatic embryogenesis is genotype-dependent in this species. Radish is self-incompatible and hybrid varieties are widely cultivated. Parental lines, however, must be maintained by labor-intensive pollination. The tissue culture method may be a useful alternative for the maintenance and proliferation of self-incompatible lines. Although shoot-tip

Acknotdedgements This research was supported by a grant

(G70580) from the Korea Ministry of Science & Technology to J.R.L. We thank Drs. Sang Soo Kwak and Haeng Soon Lee for their critical reading of this manuscript and Miss Chang Sook Kim for her assistance in its preparatiol~

References
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