Plant Cell, Tissue and Organ Culture (2005) 83: 241–244 DOI 10.1007/s11240-005-6587-3
Ó Springer 2005
Research note
Effects of growth regulators on direct flowering of isolated ginseng buds in vitro Choun-Sea Lin1,*, Chang-Tsern Chen2, Han-Wen Hsiao3 & Wei-Chin Chang4 1
Department of Biotechnology, China Institute of Technology, Taipei, Taiwan, Republic of China; 2Council of Agriculture, Executive Yuan, Republic of China; 3Institute of Bioinformatics, Taichung Healthcare and Management University, Taichung, Taiwan, Republic of China; 4Institute of Botany, Academia Sinica, Taipei, Taiwan, Republic of China (*request for offprints; E-mail:
[email protected])
Received 23 August 2004; accepted in revised form 27 April 2005
Key words: cytokinin, direct flowering, gibberellic acid, naphthaleneacetic acid, Panax ginseng
Abstract An in vitro method for obtaining gingseng inflorescences directly from explants of gingseng (Panax ginseng) is reported. Isolated shoot-buds of somatic embryo-derived plantlets ginseng were used as explants and incubated in B5 medium supplemented with 1 mg l)1 benzyladenine (BA) and 1 mg l)1 gibberellic acid (GA3). About 15% of the buds flowered directly without developing vegetative organs. Cytokinin was found to be the key factor for inducing these isolated buds to proliferate and flower, but both these processes also occurred when benzyladenine (BA) was replaced by thidiazuron (TDZ). The optimal concentration of TDZ for obtaining the best ratios of bud proliferation and total flowering was 0.1 mg l)1, while the highest number of vegetative shoots was obtained in medium supplemented with 1 mg l)1 GA3 and 0.1 mg l)1 TDZ. The explant elongated abnormally in the presence of 10 mg l)1 GA3. Although a low concentration (1 mg l)1) of NAA increased the bud proliferation ratio in the medium supplemented with 0.1 mg l)1 TDZ and 1 mg l)1 GA3, a high concentration (5 mg l)1) of NAA reduced the bud proliferation ratio and inhibited the flowering. Abbreviations: BA – benzyladenine; GA3 – gibberellic acid; NAA – naphthaleneacetic acid; TDZ – thidiazuron Season-independent in vitro flowering has been reported in ginseng (Panax ginseng C.A. Meyer) tissue culture (Chang and Hsing, 1980a; Lee et al., 1990, 1991; Lim et al., 1997; Kevers et al., 2000; Tang, 2000; Lin et al., 2003b). In most of these investigations, miniature flowers with considerable vegetative attachments formed indirectly on the regenerated plantlets (Lee et al., 1990; 1991; Lim et al., 1997; Kevers et al., 2000; Tang, 2000), although Chiang and Hsing (1980a) observed flowers that protruded directly from the shoot meristems of root-derived somatic embryos devoid of any intervening vegetative development stage. Flower or inflorescence proliferation, i.e., isolated flowers producing more similar flowers, has been described recently under defined tissue culture conditions for bamboo and ginseng (Lin et al.,
2003b). Lin et al., (2004) subsequently defined the hormonal regulation of flower proliferation in bamboo tissue culture (Lin et al. 2004). We continued this line of research in order to explore the development of the isolated ginseng shoot-buds under in vitro conditions. In this communication, a direct flowering system of isolated shoot-buds is described. The embryogenic cell line used for this study was derived from a mature ginseng root (Chang and Hsing, 1980b). The cell line was subcultured in MS medium (Murashige and Skoog, 1962) supplemented with 1 mg l)1 2,4-dichlorophenoxyacetic acid (2,4-D ) (Sigma, St. Louis, Mo.) with subculturing every 5 weeks for long-term maintenance of the somatic embryos (Chang and Hsing, 1980b). Somatic embryo-derived plantlets were cultured
242
Figure 1. Development of isolated ginseng shoot-buds. (1-1) Typical dormant buds used in all experiments (Arrowhead: dormant bud; bar = 2 mm). (1-2) Section of dormant ginseng bud showing the shoot meristems (Bar = 200 lm). (1-3) Elongation of an inflorescence after a 10-day culture in B5 medium supplemented with 1 mg l)1 BA, 1 mg l)1 GA3, and 20 g l)1 sucrose (Bar = 1 mm). (1-4) Blooming inflorescence following a 2-month culture in B5 medium supplemented with 1 mg l)1 BA, 1 mg l)1 GA3, and 20 g l)1 sucrose (Bar = 2 mm). (1-5) Expanded explant following a 2-month culture in B5 medium supplemented with 5 mg l)1 NAA, 20 g l)1 sucrose, and 2.2 g l)1 Gelrite (Bar = 2 mm). (1-6) Abnormal elongation in B5 medium supplemented with 10 mg l GA3, 20 g l)1 sucrose and 2.2 g l)1 Gelrite after 2 months (Bar = 2 mm).
in vitro in B5 medium (Gamborg et al. 1968) supplemented with 1 mg l)1 benzyladenine (BA) (Sigma), 1 mg l)1 gibberellic acid (GA3) (Sigma), 20 g l)1 sucrose, and 2.2 g l)1 Gelrite, with subculturing every 2 months (Chang and Hsing, 1980a). A large cluster of dormant buds formed at the base of each plantlet during the culture period. This cluster was separated into a number of smaller clusters, each containing three to five isolated buds, which formed the experimental material for subsequent experiments (Figure 1-1, 1-2). All cultures were incubated at 20 °C under a 16/8 h (light/dark) photoperiod with light supplied by daylight fluorescent tubes (FL-30D/29, 40 W, China Electric Co., Taipei) at an intensity of 54 lmol m)2 s)1. The basal medium consisted of B5 medium supplemented with 20 g l)1 sucrose and 2.2 g l)1 Gelrite. The growth regulators, with the exception of GA3, were added before the pH of the medium was adjusted to 5.5. The medium was then autoclaved for 15 min, cooled, and filter-sterilized GA3 was
added. Different concentrations of plant growth regulators (Sigma) were added to the basal medium to evaluate their effects on direct flowering. One cluster of isolated buds was placed on 10 ml of solid medium in a 2-cm-diameter test tube. Each treatment consisted of two to seven tubes as replicates, and for each treatment a mean value was obtained by averaging the results of three replicates. Data were scored after a 2-month culture period. Bud proliferation and flowering were defined as: bud proliferation ratio No. of buds after incubation ¼ No. of original dormant buds
flowering (% ) ¼
No. of flowering explants No. of explants
For the parameter bud proliferation, the term ‘‘number of buds’’ in the numerator includes
243 Table 1. Effects of growth regulators on isolated ginseng buds development in B5 medium TDZ (mg l)1)
Exp. 1 0 0.001 0.01 0.1 1 Exp. 2 0.1 0.1 0.1 Exp. 3 0.1 0.1 0.1
GA3 (mg l)1)
NAA (mg l)1)
Bud proliferation ratio
Flowering (%)
Direct flowering inflorescences
Indirect flowering inflorescences
Vegetative shoots
Dormancy buds
1 1 1 1 1
0 0 0 0 0
1.67b 3.38ab 2.63ab 3.87ab 4.89a
0.00c 19.00bc 37.00ab 59.67a 53.67ab
0.00a 0.11a 0.38a 0.41a 0.34a
0.00a 0.10a 0.28a 0.70a 0.87a
0.39b 0.58b 0.89ab 1.30ab 1.61a
1.28a 2.59a 1.08a 1.46a 2.07a
0 0.1 1
0 0 0
3.47a 3.51a 4.33a
20.00a 33.33a 33.33a
0.30a 0.48a 0.48a
0.23a 0.29a 0.14a
0.11b 0.13b 0.91a
2.83a 2.61a 2.80a
1 1 1
0 1 5
4.47a 7.21a 2.76a
35.00a 40.00a 0.00b
0.59a 0.93a 0.00b
0.00a 0.36a 0.00a
0.22a 1.58a 0.32a
3.66a 4.34a 2.44a
Values (means of three replicates) within a column followed by the same letter are not significantly different at p < 0.05. See text for abbreviations and definition of terms
dormant buds, inflorescences that developed directly from the explant (direct flowering), inflorescences that developed via bud-derived vegetative shoot (indirect flowering), and vegetative shoots. Histology was used to identify the development of buds (flower buds or vegetative buds). The isolated buds were fixed in FAA (formaldehyde:acetic acid:50% ethanol; 1:1:18), dehydrated in TBA (t-butyl alcohol), and embedded in paraffin wax; 12-lm thick sections were then cut and stained with 0.5% safranin-O and 0.1 % fast green. The clusters of isolated buds cultured in medium supplemented with 1 mg l)1 BA, 1 mg l)1 GA3, and 20 g l)1 sucrose for 2 months exhibited direct flowering, indirect flowering, and dormant bud and vegetative shoot development. There were no structure or morphological differences between the flowers that developed from direct or indirect inflorescences, as defined above. The direct flowering inflorescence commenced sprouting 10 days following culture initiation (Figure 1–3) and then bloomed to form miniature flowers (Figure 1–4) in subsequent subcultures. Chang and Hsing (1980a) and Lin et al. (2003b) observed direct flowering in ginseng explants cultured on medium supplemented with BA and GA3. Consequently, cytokinin has been
considered to be an important prerequisite for flowering in ginseng (Chang and Hsing, 1980a; Lee et al., 1990, 1991; Lim et al., 1997; Tang, 2000). We observed that flowering, the number of inflorescences that developed from direct flowering, and the number of inflorescences that developed from indirect flowering increased in thidiazuron (TDZ)-treated cultures (Table 1). Maximum flowering, 59.67%, was found at 0.1 mg l)1 TDZ. TDZ also promoted the number of dormant buds. As gibberellins have also been shown to be an essential element for ginseng flowering in vitro (Chang and Hsing, 1980a; Lee et al., 1990, 1991; Lim et al., 1997; Tang, 2000), we also investigated the effects of GA3 on the development of isolated buds. GA3 alone did not promote bud proliferation, and at a concentration of 10 mg l)1 GA3 the bud explant elongated abnormally (Figure 1-6). When TDZ was present in combination with GA3, percentage flowering and the number of inflorescences that developed from indirect flowering increased slightly (Table 1). An interesting observation was that GA3 was also able to increase vegetative shoot development (Table 1). During his investigations on in vitro flowering, Scorza (1982) observed that auxin was a major floral inhibitor. Lin et al. (2003a) demonstrated
244 that naphthaleneacetic acid (NAA) was a negative regulator of in vitro flowering during B. edulis shoot proliferation. In the present study, NAA used alone did not promote bud proliferation: in medium containing only 5 mg l)1 NAA as the plant growth regulator, the ginseng explant did not sprout and the base of the bud only expanded (Figure 1-5). When 1 mg l)1 NAA was combined with GA3 and TDZ in the culture medium, the bud proliferation ratio, flowering, and the number of inflorescences that developed via direct flowering increased slightly; however, at 5 mg l)1 NAA flowering did not occur (Table 1). In conclusion, we report here an in vitro seasonindependent approach for obtaining gingseng inflorescences that develop from the explants directly, without any intervening vegetative stage – i.e. direct flowering. Cytokinins (TDZ and BA) are the key components for achieving direct flowering, while NAA had a negative role in this process. The presence of GA3 in the culture medium increased both the number of inflorescences that developed via direct flowering and the flowering ratio as well as increasing the number of vegetative shoots.
Acknowledgements We thank Ms. Chen-Chen Shih and Mr. Duncan Herbert for helpful discussions that aided the preparation of this manuscript. This work was supported by grants from Academia Sinica and the National Science Council of Taiwan.
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