Springer 2005
Plant Cell, Tissue and Organ Culture (2005) 80: 157–161
Plant regeneration through somatic embryogenesis from callus cultures of Dioscorea zingiberensis Yuan Shu, Yan Ying-Cai & Lin Hong-Hui* College of Life Science, Sichuan University, Chengdu 610064, Sichuan, P.R. China (*requests for offprints: Fax: +86-028-85412571; E-mail:
[email protected]) Received 17 October 2003; accepted in revised form 7 May 2004
Key words: callus formation, somatic embryos, stem explants, yams
Abstract A new method was established for somatic embryogenesis and plant regeneration from callus cultures of Dioscorea zingiberensis C.H. Wright. Primary callus was induced by culturing stems, leaves and petioles on Murashige and Skoog (MS) medium supplemented with 0.5–2.0 mg l)1 N6-benzyladenine (BA) and 0– 2.0 mg l)1 a-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D ) for 1 month. The highest frequency (87%) of callus formation was achieved from stem explants treated with 0.5 mg l)1 BA and 2.0 mg l)1 2,4-D . Somatic embryos were obtained by subculturing embryogenic calli derived from stem explants on MS medium supplemented with 2.0–4.0 mg l)1 BA and 0–0.4 mg l)1 NAA or 2,4-D for 3 weeks. The optimum combination of 4.0 mg l)1 BA and 0.2 mg l)1 NAA promoted embryo formation on one-third of the calli. After a further month of subculture on the same medium, mature embryos were transferred to MS medium supplemented with 0–4.0 mg l)1 BA, NAA or indole-3-butyric acid (IBA) for further development of plantlets and tuber formation. Plant growth regulators had a negative effect on the development of mature embryos. Abbreviations: 2,4-D – 2,4-dichlorophenoxyacetic acid; BA – N6-benzyladenine; IBA – indole-3-butyric acid; NAA – a-naphthaleneacetic acid
The genus Dioscorea L. (yams) of about 600 species is widely distributed in the world and many of them have been used in the pharmaceutical industry. Diosgenin is a steroidal compound used as a natural source of sex and corticosteroid hormones. Early investigation showed that D. zingiberensis C.H. Wright was one of the species producing a high concentration of diosgenin. However, it has been so intensely harvested that there are only a few wild plants of D. zingiberensis left (Yongqin et al., 2003). Thus, it is crucial to explore the possibility to propagate this species in vitro. Since the 1980s, in vitro multiplication of Dioscorea species has been performed by using
explants, such as nodal cuttings (Alizadeh et al., 1998; Yongqin et al., 2003), calli (Viana and Mantell, 1989; Kohmura et al., 1995), immature leaves (Kohmura et al., 1995), cells and protoplasts (Tor et al., 1998) and microtubers (John et al., 1993). However, reports about somatic embryos derived from explants of Dioscorea species are limited (Osifo, 1988; Twyford and Mantell, 1996), and there has been no report of induction of somatic embryogenesis in D. zingiberensis. We report here a protocol to regenerate plants via somatic embryogenesis from callus from stem explants of D. zingiberensis. Seedlings of Dioscorea zingiberensis C.H. Wright were obtained from Sichuan Province,
158 China, generously donated by Dr. Tan Zhongming (College of Life Science, Sichuan University). Seedlings were cleaned under running tap water for 12 h and disinfected in 70% ethanol for 2 min, followed by 0.1% mercuric chloride for 5 min and rinsed six times with sterile water. Immature stems 5 mm long, 5 · 5 mm2 leaf sections cut from the leaf blades and petioles of 5 mm long were cultured in 250 ml Erlenmeyer flasks with 25 ml medium. The medium consisted of MS medium including MS salts, vitamins, and 2.22–8.88 lM (0.5-2.0 mg l)1) BA (Sigma, St. Louis, MO) in combination with 0–10.7 lM (0–2.0 mg l)1) NAA (Sigma) or with 0–9.05 lM (0–2.0 mg l)1) 2,4-D (Sigma), and 0.9% agar (Purified agar, Sigma) and 3% sucrose. The pH of the medium was adjusted to 5.8 with KOH or HCl prior to autoclaving for 15 min at 121 C. Explants were incubated under a 12:12 h photoperiod at 30 lmol m)2 s)1 (Coolwhite fluorescent tubes, Philips, The Netherlands) and 25 ± 1 C. The percentage of explants producing calli was determined for each treatment. Twenty explants were evaluated per treatment. The primary calli produced from explants were
monthly subcultured three times on the same medium. The embryogenic calli derived from the best treatment (stem explants with 0.5 mg l)1 BA and 2.0 mg l)1 2,4-D ) were selected and further cultured on MS medium (3% sucrose and 0.9% agar) supplemented with 8.88–17.8 lM (2.0– 4.0 mg l)1) BA and 0–2.14 lM (0–0.4 mg l)1) NAA or 0–1.81 lM (0–0.4 mg l)1) 2,4-D . In each treatment 20 calli (5 · 5 mm2) were cultured in five Erlenmeyer flasks for somatic embryogenesis. The light intensity was adjusted to 50 lmol m)2 s)1 while other conditions were maintained as mentioned above. After 3 weeks, the primary embryos were transferred along with the undifferentiated calli to the same medium for another month. Mature somatic embryos originating from the best treatment (4.0 mg l)1 BA and 0.2 mg l)1 NAA) were transferred individually to MS medium (3% sucrose and 0.9% agar) supplemented with 2.22–17.8 lM (0.5–4.0 mg l)1) BA, 2.69– 21.5 lM (0.5–4.0 mg l)1) NAA or 2.46–19.7 lM (0.5–4.0 mg l)1) IBA (Sigma). The cultures were
Table 1. Effects of BA, NAA and 2,4-D on the frequency of callus formation from stem, leaf and petiole explants of D. zingiberensisa (20 explants per treatment) Growth regulators (mg l)1) BA 0.5 0.5 0.5 0.5 1.0 1.0 1.0 1.0 2.0 2.0 2.0 2.0
0.5 0.5 0.5
NAA
Callus formation frequency (%) 2,4-D
Stem
Leaf
Petiole
0.5 1.0 2.0 0.5 1.0 2.0
Off 32 d,e 60 c 73 b 2h 15 g 27 e,f 40 d 2h 13 g 23 e,f,g 38 d 18 f,g 22 e,f,g 38 d 42 d 65 b,c 87 a
Off 25 c 17 c,d,e 13 d,e,f Off 18 c,d,e 15 c,d,e 3f Off 10 e,f 12 e,f 3f 12 e,f 20 c,d,e 37 b 18 c,d,e 55 a 23 c,d
Off Off Off Off Off 2d 3 c,d 7 a,b,c 3 c,d 8 a,b,c 10 a,b 12 a Off Off Off 2d 5 b,c,d 10 a,b
0.5 1.0 2.0 0.5 1.0 2.0 0.5 1.0 2.0
a Means within a single column followed by the same letter were not significantly different according to Ducan’s multiplication range test at the 5% level.
159 incubated under the conditions described above. All the experiments were repeated twice. All experiments were set up in a completely randomized design. Difference between means was scored with Duncan’s multiplication range test. The analysis of samples from each treatment was statistically evaluated by analysis of variance (ANOVA, p £ 0.05%) and the interactive effect of two phytohormones was assessed by two-way ANOVA. Primary calli of D. zingiberensis were initiated using stems, leaves and petioles. Table 1 shows frequencies of callus induction in relation to the types of explants and growth regulators. Auxins were essential for callus formation in all explants and 2,4-D was more efficient than NAA. Almost no callus was formed when the concentration of 2,4-D or NAA was below 0.5 mg l)1. High concentrations of auxins facilitated callus formation. Combinations of BA and 2,4-D were more favorable than 2,4-D alone. However, BA used as the sole growth regulator could not induce formation of calli. For stem as well as leaf explants, all doses of BA above 0.5 mg l)1 reduced callus formation. Stem explants formed more calli than leaf explants and petioles formed only a few calli. The highest frequency (87%) of callus formation was found on stem explants treated with 0.5 mg l)1 BA and 2.0 mg l)1 2,4-D (Table 1).
The first calli from stem explants and petiole explants consisted of small groups of transparent cells that subsequently gave rise to yellow compact calli, while the leaf-derived calli were white and loose, and grew very slowly (Yan et al., 2002). Somatic embryos appeared only on the surface of compact, yellow calli that were produced by subculturing stem-derived calli on the same medium three times. The combination of BA with auxin was essential for somatic embryogenesis. The concentration of NAA and 2,4-D strongly affected embryo formation (Table 2). Medium with BA alone only induced callus that turned green. The highest frequency (33%) of calli forming somatic embryos and averaging 40 primary embryos per callus were found in stem-derived calli on MS medium supplemented with 4.0 mg l)1 BA and 0.2 mg l)1 NAA. A significantly higher percentage of immature embryos matured if exposed to BA (4.0 mg l)1) and NAA (0.1 mg l)1) than if treated with any of the other growth regulator combinations (Table 2). The white, compact, primary embryos with a diameter of about 1 mm usually clustered together on the surface of the callus (Figure 1a and b). After 1 month of culture, the immature embryos enlarged into green ellipsoidal embryos (Figure 1c). Subsequently these developed into mature
Table 2. Effects of BA, NAA and 2,4-D on induction and conversion of somatic embryos from stem-derived callus of D. zingiberensisa (20 explants per treatment) Growth regulators (mg l)1)
BA 2.0 2.0 2.0 2.0 4.0 4.0 4.0 4.0 2.0 2.0 2.0 a
NAA
% of calli that Embryos formed embryos per callus
Number of ma- Percentage of ture embryos per immature emcallus bryos converting into mature ones (%)
7 d,e 32 a 20 b 18 b,c 5e 17 b,c 33 a 18 b,c 12 c,d 22 b 23 b
Off 1.6 3.3 2.7 0.7 3.8 4.0 2.5 1.7 3.1 1.6
2,4-D
0.1 0.2 0.4 0.1 0.2 0.4 0.1 0.2 0.4
5f 60 a 30 c,d 25 d,e 5f 10 f 40 b,c 15 e,f 10 f 15 e,f 50 a,b
Off 3c 11 b,c 11 b,c 13 b,c 38 a 10 b,c 16 b 17 b 21 b 3c
Means within a single column followed by the same letter were not significantly different according to Ducan’s multiplication range test at the 5% level.
160
Figure 1. Plant regeneration through somatic embryogenesis in Dioscorea zingiberensis. (a) Primary somatic embryos differentiated directly on the surface of the callus. Arrow One somatic embryo with distinct epidermis. Bar ¼ 850 lm; (b) primary embryos separated from the surface of the callus and several embryos clustering together. Bar ¼ 2.18 mm; (c) a mature somatic embryo showing emergence of primary root and shoot. The right part is an amplification of the left part. Left bar ¼ 927 lm; Right bar ¼ 523 lm; (d) mature embryos showing primary shoots and roots. Bar ¼ 1.35 mm; (e) two somatic embryo-derived plantlets showing a cluster of roots, shoots and inflated stem. Bar ¼ 7.00 mm; (f) a well-developed plantlet recovered from embryos. Bar ¼ 1.54 cm.
embryos with a primary shoot and root (Figures 1c and d). The mature embryos were easily dislodged from each other and the parent callus. Parallel with embryogenesis, root organogenesis also occurred on most calli, but these roots could neither form somatic embryos nor develop into plantlets. Dioscorea zingiberensis is a monocotyledonous plant. Like the zygotic embryos of D. zingiberensis, its somatic embryos did not develop into heart or torpedo-shaped embryos. Cotyledonary embryos were not observed. We merely found globular and oblong somatic embryos. To prompt elongation of roots and shoots, mature somatic embryos were cultured on media supplemented with different levels of BA, IBA and NAA for 1 month. However, embryos cultured with growth regulators did not develop more shoots and roots. Five of the mature embryos developed into plantlets with healthy roots and shoots (Figure 1e). After 2 month of culture, the plantlets showed new shoots and roots, and formed tubers (Figure 1f).
Acknowledgements This work was supported by Sichuan Science and Technology Foundation for Young Scientists. We thank Dr. Jia Yong-Jiong and Dr. Xie ming-Tang for helpful discussions and comments and Dr. Zeng Zong-Yong for assistance with the statistical analysis. We also thank Dr. Tan Zhong-Ming for providing donor plants of D. zingiberensis.
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