Plant Cell Rep (2003) 22:105–109 DOI 10.1007/s00299-003-0642-5
CELL BIOLOGY AND MORPHOGENESIS
K. P. Lee · D. W. Lee
Somatic embryogenesis and plant regeneration from seeds of wild Dicentra spectabilis (L.) LEM Received: 24 January 2003 / Revised: 10 April 2003 / Accepted: 11 April 2003 / Published online: 4 July 2003 Springer-Verlag 2003
Abstract Regeneration via somatic embryogenesis from callus was studied in Dicentra spectabilis. To obtain somatic embryogenic callus, we cultured D. spectabilis seeds on MS basal media supplemented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D). The highest percentage of embryogenic callus formation was observed on media containing 1.0 mg/l 2,4-D under dark conditions. Somatic embryogenesis was studied by transferring the callus onto MS basal medium containing different concentrations (0.0, 0.1, 0.5, 1.0, 2.0 mg/l) of KIN (kinetin) and/or BAP. Somatic embryogenesis on MS basal media with 1.0 mg/l of KIN was excellent under light conditions. Somatic embryos were rooted by transferring them to half-strength MS basal media containing 2 g/l Phytagel. About 64.2% of the somatic embryos converted to rooted plantlets, 4% showed secondary embryogenesis and 31.8% did not develop and died. Rooted plantlets showed a 46% survival rate when acclimatized ex vitro. Keywords Wild flowers · Callus · Plant regeneration · Somatic embryogenesis · Dicentra spectabilis Abbreviations BAP: 6-Benzylaminopurine · 2.4-D: 2,4-Dichlorophenoxyacetic acid · KIN: Kinetin · SEM: Scanning electron microscopy
Introduction The wild flower population in Korea includes around 170 families and 2,898 species (Kim and Hyun 1996). These are known to possess some desirable attributes, including a strong resistance to disease and the ability to endure
severe cold stress. However, there are some problems that must be overcome in order to develop domesticated blooming plants from this wild stock, namely, their dull color, short blooming time, and size. The question of whether to apply conventional breeding methods or transformation techniques for the development of commercial blooming plants from wild flowers is a pertinent one. Given that transformation techniques are widely viewed as the most effective solutions for plant breeding, we have started to investigate plant regeneration of Dicentra spectabilis L. (LEM). D. spectabilis, one of the most famous and beautiful wild flowers found in Korea, is sometimes used as a blooming plant and can be found in gardens, parks, or terraces throughout the country; it is also grown as a perennial herb. Its roots have been used in folk medicine for treating paralysis and pus and have been reported to contain coptisine, cheilantifoline, scoulerine, and protoberberine (Iwasa and Kim 1997). It bears racemes of pink and white flowers with more than ten flowers at the terminal branch of the plant. The method of propagation of this species is by seeds and in order to germinate, the seeds must endure dormancy under a cold winter period. Lazarz et al. (1982) reported in vitro propagation of D. spectabils, and plant regeneration has been reported in a few Dicentra species, such as D. peregrina (Konishi et al. 1998), but the regeneration or transformation of D. spectabilis has not been reported to date. In our laboratory, we were able to induce embryogenic callus and regenerate complete plantlets from seeds. We present here the first report of the regeneration of D. spectabilis through somatic embryogenesis.
Materials and methods Communicated by H. Lrz
Plant material and seed sterilization
K. P. Lee · D. W. Lee ()) Department of Life Science, Dongguk University, 780-714 Kyongju, Republic of Korea e-mail:
[email protected] Fax: +82-54-7702515
Seeds of Dicentra spectabilis were collected from the Forest Environment Research Station, Kyeoungsangbukdo, South Korea in May and June 2001. The seeds still encased by green-colored seed coats were rinsed in 70% ethanol for 1 min, sterilized in 1.5%
106 Table 1 The effects of concentrations of 2,4-D on embryogenic callus formation from seeds of Dicentra spectabilis (L.) during 120 days of culture. Data correspond to the average results of two independently repeated experiments
2,4-D (mg/l)
Number of seeds
Frequency of seeds forming embryogenic callus (%)
0.0 0.1 0.5 1.0 2.0
144 120 120 120 120
0 2.50€0.83a 5.83€2.50 90.84€5.84 16.66€4.99
a
Mean € standard error
sodium hypochlorite solution for 5 min, and then rinsed three times for 10 min with sterile distilled water. We then removed the seed coats cut the sterilized mature seeds (at the black-colored stage) in half, and cultured the latter on callus induction medium. Embryogenic callus induction from seeds For the induction of embryogenic callus, the sterilized seeds were cultured on MS (Murashige and Skoog 1962) basal medium containing 3% sucrose, 0.4% Phytagel, and 0.1% casamic acid supplemented with 2,4-D (0.1, 0.5, 1.0 or 2.0 mg/l), pH 5.7, at 24€2C in the dark. The induction index of the callus was calculated as described previously (Cheon and Lee 1999) after 120 days of culture. Low-temperature treatment of seeds To shorten the callus induction periods, we first exposed the sterilized seeds to 4C for 10, 20, 30, or 60 days (Norton 1985) and then cultured them on MS basal medium supplemented with 3% sucrose, 0.4% Phytagel, 0.1% casamic acid and 1.0 mg/l 2,4-D. Somatic embryogenesis maturation Embryogenic calli, with globular embryos induced on induction medium containing 1.0 mg/l 2,4-D, were cultured on MS basal medium or on MS basal medium supplemented with KIN (0.1, 0.5, 1.0 or 2.0 mg/l), BAP (0.1, 0.5, 1.0 or 2.0 mg/l), and 2,4-D (0.1 mg/ l) in combination with KIN (1.0 or 2.0 mg/l) or BAP (1.0 or 2.0 mg/ l). All cultures were maintained in a culture room at 24€2C under a 16/8-h (light/dark) photoperiod with light supplied by white fluorescent lamps at an intensity of 24 m Em-2 s-1. Somatic embryos—germination and regeneration Cotyledon somatic embryos at the cotyledonary stage were collected from the previous experiments and cultured on the same medium for 4 more weeks. To develop roots, the plantlets were subcultured on 1/2-strength MS basal medium supplemented with 15 g/l sucrose and 2, 4 or 6 g/l Phytagel and then transferred exvitro.
Results and discussion After 90 days of culture, the initial callus was induced in the seed explants on the cut surfaces irrespective of the medium; callus subsequently developed and covered the entire plant surface. There was no evidence of callus formation when explants were cultured in media without auxin. The medium containing 1.0 mg/l 2,4-D proved to be very effective at inducing profuse callusing (Table 1). The primary callus was yellowish-white but this greened
rapidly and turned into a yellowish-brown friable callus upon subsequent culture. After 4 weeks on callus induction media (MS with 1 mg/l 2,4-D), the yellowish-brown calli derived from seed explants (Fig. 1C) had developed into transparent white clumps of embryogenic calli. The highest efficiency (90.84%) of embryogenic calli induction (Table 1) was obtained on MS media supplemented with 1 mg/l 2,4-D. Embryogenic calli developed into globular-, heart-, and torpedo-shaped somatic embryos (Fig. 1-A, B, D, E) on the induction medium, with globular-shaped embryos appearing after 120 days. The size of initial globular embryo was very small and completely globular in shape (Fig. 1A). Embryogenic calli containing the globularembryo type were used for further maturation experiments. The globular somatic embryos turned into heartand torpedo- shaped somatic embryos. Torpedo-shaped somatic embryos did not develop completely into decotyledonary-shaped ones (Fig. 1B, F), however they did not develop abnormally but as complete plantlets (Fig. 1G, H). The fastest initial callus induction was observed at 13 days of culture following 30 days of cold treatment. The callus induction rate was also increased after 30 days of cold treatment (Table 2). This result seems to be related to seed propagation endurance under cold winter. The beneficial effect of the presence of a cytokinin in the culture medium for stimulating somatic embryogenesis from the legume calli was demonstrated previously by Mroginski and Kartha (1984). In preliminary experiments, we found that both KIN and BAP had the ability to induce somatic embryogenesis. Consequently, we investigated the optimal concentration of KIN or BAP required to induce somatic embryogenesis and found that 1.0 mg/l KIN or 2.0 mg/l BAP produced the highest frequency of globular embryos producing torpedo- and cotyledonary somatic embryos (Table 3). It has been reported that it is the combination of low levels of auxin and high levels of cytokinins (BAP, KIN), when introduced to soybean (Shoemaker et al. 1991), Fig. 1A–H Somatic embryogenesis in Dicentra spectabilis L. (LEM). A SEM of the globular-stage somatic embryo developed from surface cells of seeds, B SEM of dipole-developed cotyledonary embryo, C callus induced from seed of D. spectabilis after 90 days of culture, D globular embryos (arrows) after 120 days of culture, E torpedo embryos on the embryogenic callus, F cotyledonary embryo (arrow), G leaf development (arrow) from somatic embryo, H development of leaves and stems by somatic embryos. Bar: 100 mm (B), 250 mm (C), 300 mm (D), 1.2 mm (E), 1.5 mm (F), 1.0 mm (G), 1.5 mm (H)
107
108 Table 2 The effects of low temperature (4C) treatment on callus formation from seed. Data correspond to the average results of two independently repeated experiments Periods of low temperature treatments (days)
Number of Seeds
Periods of initial callus formation after culturing (days)
Frequency of callus induction from seeds germinated on MS medium supplemented with 1 mg/l 2,4-D after 60 days of culture (%)
10 20 30 60
80 80 80 80
45 28 13 30
4.38€1.88a 26.88€3.13 81.88€1.88 45.00€3.75
a
Mean € standard error
Table 3 The effects of plant growth regulators on somatic embryo maturation. Embryogenic calli with globular embryos were transferred onto different media for 1 month. Data correspond to the average results of two independently repeated experiments Maturation media
Number of globular embryos
Frequency of globular embryos producing torpedo and cotyledon somatic embryos (%)
MS MS MS MS MS MS MS MS MS MS MS MS MS
102 96 114 126 117 132 120 120 117 174 132 120 129
0 0 0.75€1.54a 100.00€0.00 31.42€2.26 36.68€3.38 48.94€2.64 78.40€4.44 95.88€1.86 22.82€0.89 26.93€1.12 11.57€0.38 29.44€3.32
a
+ + + + + + + + + + + +
0.1 mg/l KIN 0.5 mg/l KIN 1.0 mg/l KIN 2.0 mg/l KIN 0.1 mg/l BAP 0.5 mg/l BAP 1.0 mg/l BAP BAP 2.0 mg/l 0.1 mg/l 2,4-D 0.1 mg/l 2,4-D 0.1 mg/l 2,4-D 0.1 mg/l 2,4-D
+ + + +
1.0 2.0 1.0 2.0
mg/l mg/l mg/l mg/l
KIN KIN BAP BAP
Mean € standard error
Table 4 The effect of Phytagel on root development. Data corresponds to the average result from two independent repeated experiments Phytagel (mg/l)
Number of plantlets
Frequency of roots developed on plantlet via somatic embryogenesis (%)
2.0 4.0 6.0
145 138 151
64.2€2.1a 22.5€5.8 37.5€11.0
a
Mean € standard error
Fig. 2A, B Plant regeneration of D. spectabilis L. (LEM). A Plant obtained by rooting a regenerated shoot after a 6month culture on MS medium, B plants growing in soil containing 50% vermiculate, transferred to ex vitro after 6 months in vitro culture. Bar: 1.0 cm (A), 1.0 cm (B)
109
Aesculus hippocastanum (Radojevic 1988), Typhonium trilobatum (Das et al. 1999) and Cephaelis ipecacuanha (Rout et al. 2000), that results in somatic embryogenesis. For this reason, we also investigated the combined effects of 2,4-D and cytokinins. Media containing a combination of auxin and cytokinins, namely 2,4-D (0.1 mg/l) and KIN (1.0, 2.0 mg/l) or 2,4-D (0.1 mg/l) and BAP (1.0, 2.0 mg/ l), showed a decreased frequency of somatic embryogenic calli than medium supplemented with auxin but excluding cytokinins (Table 3). In our experiments, MS basal medium containing 1.0 mg/l KIN was the most efficacious for inducing somatic embryogenesis. Cotyledon somatic embryos at the cotyledonary stage were developed on the media (though containing 1.0 mg/l KIN) used in the previous experiments after 4 weeks of culture. Compared with medium supplemented with 1.0 mg/l KIN, one supplemented with 2.0 mg/l BAP resulted in nearly the same high efficiency of germination. Somatic embryos developed into normal plantlets on media containing 1.0 mg/l of KIN, whereas, on media containing 2.0 mg/l of BAP, they developed into abnormal plantlets. Mohiuddin et al. (1998) also reported that a high concentration (2.0 mg/l) of BAP induced abnormal plantlets in Cucumis melo L. The best result for root development into plantlets was observed on 1/2-strength MS basal medium supplemented with 15 g/l sucrose and 2 g/l Phytagel (Table 4). This indicated that the in vitro formation of initial roots on D. spectabilis was affected by the strength of the solidifying agent. Following root development, the normal plantlets were subcultured for 30 days in a growth bottle containing the 1/2-strength MS medium (Fig. 2A) and then transferred ex vitro (Fig. 2B). Rooted plantlets showed 46% survival when acclimatized ex vitro. In conclusion, our investigation demonstrates the successful regeneration through somatic embryogenesis of D. spectabilis by in vitro culture of the seed explants under defined environmental conditions.
Acknowledgements This investigation was supported by the Dongguk University Research Fund
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