Plant Cell, Tissue and Organ Culture 79: 341–345, 2004. 2004 Kluwer Academic Publishers. Printed in the Netherlands.
341
Research Note
Indirect somatic embryogenesis and plant regeneration from leaf and internode explants of Paulownia elongata Zeliha Ipekci1,* & Nermin Gozukirmizi1,2 1
TUBITAK, Research Institute for Genetic Engineering and Biotechnology (RIGEB), P.O. Box 21, 41470, Gebze-Kocaeli/Turkey; 2Istanbul University, Faculty of Science, Department of Molecular Biology and Genetics, 34459, Vezneciler-Istanbul/Turkey (*requests for offprints; Fax: +90-262-646-39-29; E-mail:
[email protected]) Received 31 January 2003; accepted in revised form 22 October 2003
Key words: embryogenic callus, kinetin, thidiazuron
Abstract Several plant growth regulators BA, TDZ, 2,4-D and Kn were tested alone or in combination for their capacity to induce indirect somatic embryogenesis from leaf and internode explants of Paulownia elongata. Calli were produced when leaf explants were cultured on Murashige and Skoog (MS) medium containing 3% sucrose, 0.4% phytagel, 4 mg l)1 TDZ and 0.1 mg l)1 Kn after 3 weeks and the initiation rate was 54.1%. After subculturing on the same medium, embryos at various developmental stages (globular, heart and torpedo shaped) were transferred for maturation onto MS medium supplemented with 3% sucrose, 0.4% phytagel, 0.1 mg l)1 TDZ, 1 mg l)1 Kn and 2 mM glutamine. An average of 50.7 somatic embryos were obtained from 100 mg of embryogenic callus after 4 weeks at high frequency (64.7%). Afterward, mature somatic embryos were isolated and cultured on hormone-free MS medium for germination (80%) and development into plantlets. Plantlets were transferred to pots with a mixture of peat and perlite in a 3:1 ratio and showed a survival rate of 70–80%. Plantlets regenerated by this procedure were morphologically identical to the donor material and developed normally in the greenhouse. Abbreviations: BA – 6-benzyladenine; 2,4-D – 2,4-dichlorophenoxyacetic acid; Kn – kinetin; MS – Murashige and Skoog (1962) medium; NAA – naphthalenacetic acid; TDZ – N-phenyl-N¢1,2,3-thidiazurol-5-ylurea
Paulownia is an economically important genus in the family Scrophulariaceae. It is native to China, and has been introduced in Japan, Australia, Brazil, Europe and the United States. It is characterized by an attractive growth habit and light wood. Its tolerance to drought and soil extremes makes it important commercially for use in reclamation of surface-mined land (Zhu et al., 1986). As a genus, Paulownia is receiving increased attention as a fast growing, short-rotation woody crop plant. Paulownia spp. is propagated by seeds and seedling root cuttings, with the latter being
used more often (Burger et al., 1985). Propagation by stem or root cuttings is important for producing clonal planting stock. Stem cuttings are generally more difficult to propagate than root cuttings. Therefore, development of an efficient vegetative propagation is an essential goal for Paulownia clonal forestry and has many advantages over seedling production. In vitro regeneration was achieved by organogenesis from various explant sources for several species and two hybrids of Paulownia (Ni and Ma, 1982; Marcotrigiano and Stimart, 1983; Chang and Donald, 1992;
342 Bergmann and Moon, 1997; Bergmann, 1998; Ipekci et al., 2001). Though plant regeneration via direct and indirect organogenesis from various explant sources of Paulownia species has been reported, there is only one report to date on indirect somatic embryogenesis from Paulownia tomentosa (Radojevic, 1979) and recently one report for direct somatic embryo formation from Paulownia elongata (Ipekci and Gozukirmizi, 2003). Somatic embryogenesis is a powerful tool for improvement of forest trees and is the in vitro plant regeneration system of choice for woody plants and it is regulated by an interaction of phytohormones, mainly auxins and cytokinins (Komamine et al., 1992). Although auxins are the best studied inducers for obtaining embryogenic cells, they are certainly not unique in the ability to mediate the transition to embryogenic cells. TDZ, a substituted phenylurea has stimulated induction of somatic embryogenesis in a variety of plant species and several woody species (Chevreau et al., 1989). We have attempted to further improve indirect somatic embryogenesis using TDZ with different concentrations and combinations of BA, 2,4-D or Kn. Leaf (1 cm2) and internodal segments (0.5 cm) of Paulownia elongata that served as explants were harvested from in vitro regenerated plantlets. These had originated from nodal segments of 3month old seedlings cultured on MS (Murashige and Skoog, 1962) medium supplemented with 0.1 mg l)1 NAA and 1 mg l)1 BA (Ipekci et al., 2001). The basal medium for all experiments consisted of MS mineral nutrients and vitamins with 30 g l)1 sucrose and 0.4% phytagel and plant growth regulators (TDZ, 2,4-D , BA, Kn) either alone or in combination. The pH of the medium was adjusted to 5.7 prior to autoclaving at 121 C for 20 min. Explants were incubated on sterile media in Petri dishes (100 · 15 mm) containing 25 ml of medium (6–8 explants per Petri dish) sealed with parafilm and maintained at 25 C in a growth chamber with 30 lmol m)2 s)1 illumination from cool-white fluorescent lamps and a 16-h photoperiod. For callus induction, four growth regulator combinations of 1 mg l)1 BA with 1–4 mg l)1 TDZ (MS1–MS4), four growth regulator combinations of 0.1 mg l)1 BA with 1–4 mg l)1 2,4-D (MS5–MS8), five combinations of 0.1 mg l)1 Kn with 1–5 mg l)1 TDZ (MS9–MS13) and four
combinations of 2.2 mg l)1 TDZ with 0.5– 2 mg l)1 Kn (MS14–MS17) were tested. After 3 weeks of culture, the frequency of explants producing calli was scored. Subsequently, the induced calli were detached from the explants and subcultured onto each fresh medium for long term maintenance. Callus tissues initiated from leaf and internode explants cultured on MS1–MS17 medium with somatic embryos were transferred onto embryo development media consisting of MS basal medium with 2 mM glutamine and 0.1 mg l)1 2,4-D with 0.1–1 mg l)1 BA (MSE1–MSE3), 0.1 mg l)1 TDZ with 0.1–1 mg l)1 BA (MSE4– MSE6) or 0.1 mg l)1 TDZ with 0.1–1 mg l)1 Kn (MSE7–MSE9) 3 weeks after culture initiation. The frequency of calli producing somatic embryos was recorded as the number of somatic embryos per gram callus 1 month after transfer to embryo development media. Embryogenic callus pieces with globular, heart-shaped and torpedo stage embryos were transferred to MS medium supplemented with 2 mM glutamine and four different concentrations of 0.1–1 mg l)1 Kn (MSE11–MSE14) or three different concentrations of 0.1–1 mg l)1 BA (MSE15–MSE17) for further development of embryos. Alternatively, embryos were cultured on MS medium without any plant growth regulators (MSE18). Plantlets were subcultured in GA-7 Magenta vessels (Sigma) (four plantlets in each vessel) containing 25 ml of each MSE11-MSE18 media. After 2 weeks of plant development, the frequency of conversion of somatic embryos into plantlets was calculated. Plantlets with well-developed roots were transferred to pots with an autoclaved mixture of peat and perlite in a 3:1 ratio. Experiments were set up in a completely randomized design. Each treatment consisted of 30 explants or callus pieces and each experiment was carried out three times. The data shown represent the mean ±SE of three independent experiments. ANOVA was performed on the results of each experiment and the data were analyzed using Duncan’s multiple range test (p < 0.05). Callus initiation was observed from the ends of leaf and internode explants of Paulownia elongata within 1 week on all media tested. Callus covered the entire surface of the explants in 3 weeks. The frequency of callus proliferation from leaf and internode explants was greater when cultured on media with both Kn and TDZ (Table 1). Exposure
343 Table 1. Effect of MS media with various concentrations and combinations of TDZ, 2,4-D , BA and Kn on callus induction in leaf and internode explants of Paulownia elongata after 3 weeks of culturea Percentage of explants forming callus (%)b
Media MS + growth regulator (mg l)1)
MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 MS9 MS10 MS11 MS12 MS13 MS14 MS15 MS16 MS17 a
BA
TDZ
1 1 1 1 0.1 0.1 0.1 0.1
1 2 3 4
2,4-D
Kn
Leaf
0.1 0.1 0.1 0.1 0.1 0.5 1 1.5 2
12.5 20.8 29.3 26.8 20.8 10.8 29.1 25.0 35.3 37.5 45.8 54.1 50.3 33.1 34.5 33.3 29.1
1 2 3 4 1 2 3 4 5 2.2 2.2 2.2 2.2
Internode ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.2ab 0.3bc 0.5de 0.4de 0.3bc 0.2ab 0.5bc 0.4bc 0.6de 0.6fg 0.7fg 0.8h 0.7fg 0.1de 0.5fg 0.5de 0.4bc
4.1 ± 0.06a 12.5 ± 0.2ab 12.5 ± 0.2ab 13.6 ± 0.2ab 10.6 ± 0.2ab 4.1 ± 0.06a 12.5 ± 0.2ab 15.6 ± 0.2bc 20.8 ± 0.3ab 20.8 ± 0.3bc 21.6 ± 0.3bc 25.0 ± 0.4bc 23.6 ± 0.3ab 16.6 ± 0.2bc 18.5 ± 0.3ab 18.5 ± 0.3ab 19.1 ± 0.3a
Means followed by the same letters are not significantly different at p < 0.05 according to Duncan multiple range test. 30 replicates/treatment; experiment carried out thrice.
b
to a high concentration (4 mg l)1) of TDZ was more effective than exposure to a low level (1 mg l)1) of TDZ. After 3 weeks of culture on MS medium supplemented with 0.1 mg l)1 Kn and 4 mg l)1 TDZ (MS12) the frequency of callus formation was higher from leaf explants (54.1%) (Table 1) than from internode explants (25%) on the same medium (Table 1). After 2–3 weeks of subculture, the friable calli differentiated into white embryogenic clumps in medium supplemented with 2 mM glutamine and various concentrations and combinations of BA, TDZ, 2,4-D and Kn. Initially, most of the embryogenic cultures showed a high degree of friability with many yellowish green globular structures. Subsequently, about 65% of embryogenic calli from leaf explants developed numerous globular, heart-shaped and torpedo stage embryos when cultured on MS medium supplemented with 0.1 mg l)1 TDZ and 1 mg l)1 Kn (MSE9), with 50.7 somatic embryos being produced per 100 mg of callus (Table 2) (Figure 1a, b). Only 37.5% of calli from internode explants formed embryos on the same medium (Table 2).
Embryo formation rate was generally higher when TDZ was combined with Kn rather than with BA. Increasing the Kn concentration from 0.1 to 1 mg l)1 led to a greater percentage of the calli forming embryos. After several subculture cycles on MS1–MS17 somatic embryos ceased to develop from callus. Interest in the diphenylurea TDZ has been growing in recent years due to its effectiveness in regulation of morphogenesis. TDZ induces a wide array of physiological effects and has been found to be used in a wide range of concentrations from 2 to 20 mg l)1 in different plant species. Malik and Saxena (1992), first reported the development of regenerative structures from different tissues of intact seedlings cultured on media containing TDZ. More recent findings indicate that this regeneration may be linked to TDZ-induced accumulation of several minerals including sodium, copper and zinc (Murch and Saxena, 1997). The germination (Figure 1c) rate of somatic embryos from 45 leaf explants was 80% on MS medium lacking plant growth regulator. From 100 mg of embryogenic callus, an average of 65–70 plantlets were obtained. The plantlets were accli-
344 Table 2. Effect of MS medium with various concentrations and combinations of plant growth regulators on development of somatic embryos and number of embryos per culture derived from leaf and internode explants after one month of callus culturea Frequency (%) of calli undergoing somatic embryogenesis
Media MS + 2 mM glutamine + plant growth regulator (mg l)1) BA MSE1 MSE2 MSE3 MSE4 MSE5 MSE6 MSE7 MSE8 MSE9 a
0.1 0.5 1 0.1 0.5 1
TDZ
2,4-D
Kn
Leaf
0.1 0.5 1
29.4 29.4 31.1 33.5 17.6 14.7 37.5 44.1 64.7
0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Internode ± ± ± ± ± ± ± ± ±
0.8cd 0.8cd 1.1cd 1.1cd 0.2ab 0.2a 1.1cd 1.2e 1.9e
20.5 17.6 14.7 23.5 20.5 17.6 14.7 23.5 37.5
± ± ± ± ± ± ± ± ±
0.7ab 0.2ab 0.2a 0.6cd 0.7ab 0.2ab 0.2a 0.6cd 1.1cd
No. of somatic embryos/cultureb
Leaf 11.8 12.6 23.4 23.4 7.6 23.4 23.4 26.4 50.7
Internode ± ± ± ± ± ± ± ± ±
0.8b 0.8b 0.6c 0.6c 0.7b 0.6c 0.6a 0.6c 0.2d
7.6 8.6 13.4 14.8 7.8 8.6 13.4 14.8 19.4
± ± ± ± ± ± ± ± ±
0.7b 0.7b 0.5c 0.6c 0.7b 0.7c 0.2a 0.2c 0.6d
Means followed by the same letters are not significantly different at p < 0.05 according to Duncan multiple range test. 30 callus explants/treatment; experiment carried out thrice.
b
Figure 1. Indirect somatic embryogenesis of Paulownia elongata: (a) somatic embryo (arrows) production from embryogenic callus after one month following transfer to MS + 0.1 mg l)1 TDZ + 1 mg l)1 Kn (bar ¼ 5 mM) (b) somatic embryos at various stages of in vitro development (bar ¼ 5 mM) (c) germinated somatic embryo following transfer to MS medium lacking any plant growth regulator (bar ¼ 10 mM) (d) acclimatized plantlets from both leaf and internode-derived somatic embryos (bar ¼ 10 mM).
matized for 2 months in pots in a controlled greenhouse (Figure 1d). Of the 42 plantlets that were acclimatized, 30 survived and grew well afterwards. No variation was observed in the morphology of the plantlets.
Acknowledgement This work was supported by the Research Foundation of the Istanbul University, Project No. T745/251099.
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