Efficient plant regeneration through somatic embryogenesis from
callus cultures of
Oncidium
(Orchidaceae)
Jen-Tsung Chen, Wei-Chin Chang *
Institute of Botany,Academia Sinica,Taipei,Taiwan,115People’s Republic of China
Received 6 June 2000; received in revised form 9 August 2000; accepted 9 August 2000
Abstract
An efficient method was established for high frequency somatic embryogenesis and plant regeneration from callus cultures of a hybrid of sympodial orchid (Oncidium‘Gower Ramsey’). Compact and yellow – white embryogenic calli formed from root tips and cut ends of stem and leaf segments on 1/2 MS [11] basal medium supplemented with 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea (TDZ, 0.1 – 3 mg/l), 2,4-dichlorophenoxyacetic acid (2,4-D, 3 – 10 mg/l) and peptone (1 g/l) for 4 – 7 weeks. Embryogenic callus was maintained by subculture on the same medium for callus induction and proliferated 2 – 4 times (fresh weight) in 1 month. Initiation of somatic embryogenesis and development up to the protocorm-like-bodies (PLBs) from callus cultures was achieved on hormone-free basal medium. Regenerants were recovered from somatic embryos (SEs) after transfer to the same medium and showed normal development. The optimized protocol required about 12 – 14 weeks from the initiation of callus to the plantlet formation. Generally, the frequency of embryo formation of root-derived callus was higher than stem- and leaf-derived calli. Combinations of naphthaleneacetic acid (NAA) and TDZ significantly promoted embryo formation from callus cultures. The high-frequency (93.8%) somatic embryogenesis and an average of 29.1 SEs per callus (3×3 mm2) was found in root-derived callus
on a basal medium supplemented with 0.1 mg/l NAA and 3 mg/l TDZ. Almost all the SEs converted and the plantlets grew well with an almost 100% survival rate when potted in sphagnum moss and acclimatized in the greenhouse. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Callus; Oncidium; Somatic embryogenesis
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1. Introduction
A wide range of attractive hybrids, varieties or cultivars of sympodial orchids of the genusOncid
-ium (Orchidaceae) has become economically im-portant, mainly, for use in cut-flower and potted-plant industries. The need for mass propa-gation of selected elite genotypes has led to the development of several in vitro methods [1] includ-ing culture of flower stalk-tips [2], apical-shoot explants [3,4], root tips [5], apical buds [6], flower stalk buds [7] and shoot-tips [8]. Limited plantlets were usually obtained from these explants through protocorm-like bodies (PLBs) from intermediate
callus proliferated on underfined media supple-mented with natural stuff such as banana ho-mogenate, coconut milk or tomato juice. Long term subcultures of these calli and their totipo-tency were not documented.
We have previously defined a reliable protocol for inducing direct somatic embryogenesis and subsequent plant regeneration for cultured leaf tissue ofO. ‘Gower Ramsey’ on a defined medium [9], as well as the effects of auxins and cytokinins on somatic embryogenesis [10]. Further trials by using explants of roots, stems and young leaves led to the establishment of subculturable totipo-tent calli. This present communication described requirements for callus induction, proliferation and subsequent plant regeneration via somatic embryogenesis from vegetative tissues of O. ‘Gower Ramsey’.
* Corresponding author. Tel.: +886-2-27899590, ext. 120; fax:
+886-2-27827954.
E-mail address:[email protected] (W.-C. Chang).
2. Materials and methods
Nine-month old in vitro grown donor plantlets ofOncidium ‘Gower Ramsey’ were obtained from flower stalk bud-derived PLBs [9], were generously supplied by Dr Fure-Chyi Chen (National Ping-tung University of Science and Technology, Tai-wan). Stem internodes of 5 mm, leaves of 2 – 4 cm and 5 – 7 cm, and root tips of 1 cm in length were used as explants for callusing. Explants were placed on the surface of basal medium with 1/2 strength macro- and micro-elements of Murashige and Skoog [11] supplemented with (mg/l), myo-inositol (100); niacin (0.5); pyridoxine HCl (0.5); thiamine HCl (0.1); glycine (2.0); peptone (1000); NaH2PO4 (170); sucrose (20 000); and Gelrite™ (2200). Plant growth regulators were added prior to autoclaving as optional additives according to the experimental objectives. The pH of the media was adjusted to 5.2 with 1 N KOH or HCl prior to autoclaving for 15 min at 121°C. Explants were incubated in 20×150 mm culture tubes under a 16:8 h photoperiod at 28 – 36mmole m−2s−1 (day-light fluorescent tubes FL-30D/29, 40 W, China Electric Co., Taipei) and 2691°C. Twenty repli-cates were taken for each. Four explants were planted in each culture tube. Observations were made after 2 months of growth. Difference be-tween means was scored with Duncan’s multiple range test [12].
Callus lines were usually subcultured on the original media ever 2 months and were maintained in the same culture condition as mentioned above. Cultures were examined and photographed with a stereozoom microscope (SZH, Olympus). Scan-ning electron micrographs were taken (DSM-950, Carl Zeiss) of tissues fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h at 4°C, dehydrated in ethanol [13], critical point dryer (HCP-2, Hitachi), and coated with gold in an ion coater (IB-2, Giko Engineering Co.).
3. Results
3.1. Initiation and subculture of embryogenic callus
Preliminary experiments with root segments showed that only root tips formed callus in all treatments with combinations of TDZ and 2,4-D
on 1/2 MS-basal medium. Thus, only the segments containing root tips with meristems were used as root explants for callus induction. Between 4 and 7 weeks after plating, root tips of the cultured root segments (Fig. 1a), cut surfaces of stem segments of (Fig. 1b) and young leaves (2 – 4 cm in length)
(Fig. 1c) exhibited embryogenic calli. Explants from older leaves (5 – 7 cm in length) did not formed any visible callus. Also, callusing only occurred in explants maintained devoid of light. The first outgrowths from the explants consisted
Fig. 1. Callus initiation and somatic embryogenesis ofOncid
Table 1
Effects of 2,4-D and TDZ on callus induction and proliferation from leaf, stem and root explants ofOncidiumGower Ramseya
2,4-D TDZ Leaf Stem Root
% Callusing Proliferation rate % Callusing Proliferation rate % Callusing Proliferation rate
0 a – 0 a
aThe cultures were kept in darkness at 2691°C. Data were scored after 2 months culture for callus induction or callus
proliferation. In each treatment 20 replicates (explants) were cultured in 5 culture tubes for callus induction or callus proliferation. Means of 20 replicates with the same letters are not significantly different as determined by Duncan’s multiple range test (PB0.05) [12]. Callus proliferation rate was calculated by dividing the final fresh wt.
of small groups of transient cells, and subse-quently gave rise primarily to yellow – white, com-pact, organized and embryogenic callus with a smooth surface (Fig. 1d). Table 1 shows frequen-cies of callus induction in relation to the types of explants and growth regulator treatments. The highest percentage of callus induction and the best growth occurred in root explants. Combinations of TDZ and 2,4-D were essential for callusing of the explants of leaves and roots (Table 1). Combi-nations of 3 mg/l TDZ with either 3 or 10 mg/l 2,4-D favored callusing from root tips, while lesser dosages of TDZ and 2,4-D were required for callusing from stem explants. In the presence of TDZ, ranging from 0.3 to 3 mg/l, a higher dosage (10 mg/l) 2,4-D was required for callusing from explants of young leaves of 2 – 4 cm in length. No difference was found on the proliferation rates of the subcultured calli derived from three kinds of explants. These compact and organized callus tis-sues maintained their embryogenic potential for more than 1 year when subculture culture in the original medium.
3.2. Embryo formation
respec-tively. The optimum combination of 0.1 – 1 mg/l NAA and 0.3 – 3 mg/l TDZ significantly promoted embryo formation in most callus lines. The highest frequency of somatic embryogenesis and average number of embryos per callus are 93.8%, 29.1 (R8); 25.0%, 7.5 (R12) (Table 2); 68.8%, 19.0 (S7); 18.8%, 18.3 (S8); 0% 0 (S9) (Table 3); 0%, 0 (L10); 37.5%, 7.0 (L11); 31.3%, 16.0 (L12) (Table 4), re-spectively. The highest frequency (93.8%) of so-matic embryogenesis and average 29.1 embryos per callus were found in root-derived callus on a basal medium supplemented with 0.1 mg/l NAA and 0.3 – 3 mg/l TDZ (Table 2). However, two callus lines formed no embryos in all NAA and TDZ combinations. No morphological differences were found among the embryos of callus lines.
3.3. Scanning electron microscopy obser6ation on
embryo formation
Somatic embryos emerged from the outer cell layer of callus, and apparently started to form a single cell (Fig. 2a). As the differentiation pro-gressed, the embryos started to initiate from the region further from the surface of callus (Fig. 2a,b). These embryos were composed of embryonic cells that were much smaller than cells of callus (Fig. 2b); they expanded sequentially, formed a protrusion and turned into PLBs eventually (Fig.
2c). The second sheath-leave develops from the shoot apex opposite the first organ (Fig. 2d).
3.4. Plant regeneration and con6ersion
After 2 – 3 weeks culture on the same medium for embryos induction, most embryos expanded se-quentially and germinated into PLBs with a pro-trusion in the anterior site and numerous absorbing hairs in the posterior region (Fig. 3a). The protrusion region developed into the first sheath-leave and the second sheath-leave formed from the shoot apex opposite the first organ (Fig. 3b). These two-sheath-leaves PLBs enlarged and elongated sequentially (Fig. 3c), and the subse-quent true leaves differentiate in an alternating sequence (Fig. 3d). At maturity, the basal regions of the embryo-derived PLBs contracted and the whole structure was easily dislodged from each other and the parent callus. Almost all the em-bryos/PLBs placed on hormone-free basal medium, formed individual plantlets with both roots and shoots within 2 – 3 weeks (Fig. 3e). All regenerants about 3 – 4 cm in height with five to six leaves and three to four roots were then potted in sphagnum moss and acclimatized in the green-house. These plants all grew well with an almost 100% survival rate and formed pseudobulbs after 3 – 4 months of culture (Fig. 3f).
Table 2
Effects of NAA and TDZ on somatic embryogenesis of two root callus lines ofOncidiumGower Ramsey. Data were recorded after 6 weeks culturea
Line R8 Line R12
NAA mg/l TDZ
Embryos/ callus % forming embryos
% forming embryos Embryos/callus
0 37.5 ab 8.3 0 c 0
aCultures were kept at 2691°C under a 16:8 h photoperiod at 28–36
mmole m−2s−1(daylight fluoresecent tube FL-30D/29,
40 W, China Electric Co., Taipei). Means of 16 callus explants (3×3 mm2) with the same letters are not significantly different at
Table 3
Effects of NAA and TDZ on somatic embryogenesis from three stem-derived callus lines ofOncidiumGower Ramsey. Data were recorded after 6 weeks culturea
NAA mg/l TDZ Line S7 Line S8
% Forming embryos Embryos/callus % Forming embryos Embryos/ Callus
18.8 ab 15.0
aCultures were kept at 2691°C under a 16:8 h photoperiod at 28–36
mmole m−2s−1(daylight fluoresecent tube FL-30D/29,
40 W, China Electric Co., Taipei). Means of 16 callus explants (3×3 mm2) with the same letters are not significantly different at
PB0.05 after Duncan [12].
4. Discussion
Explants of Oncidium spp. comprised mainly of floral tissues and root tips that when cultured usually proliferate to form PLBs that eventually go on to form plantlets [2 – 5,7,8,14]. Callus ob-tained from root tips of Oncidium6aricosumin an
undefined medium had outgrowths reminiscent of
malformed roots and slow growth was observed [6]. This root tip-derived callus was hard to be subcultured and had low regeneration capacity. A few PLBs formed from these calli and developed into plantlets. As we presented here, the subcultur-able calli derived from root, stem and also leaf segments of Oncidium ’Gower Ramsey’ have strong regeneration ability and could form somatic
Table 4
Effects of NAA and TDZ on somatic embryogenesis from three leaf callus lines ofOncidiumGower Ramsaey. Data were recorded after 6 week culturea
TDZ Line L11
NAA Line L12
Embryos/ callus % forming embryos
% forming embryos Embryos/callus
18.8 ab
aCultures were kept at 2691°C under a 16:8 h photoperiod at 28–36mmole m−2s−1(daylight fluoresecent tube FL-30D/29,
40 W, China Electric Co., Taipei). Means of 16 callus explants (3×3 mm2) with the same letters are not significantly different at
Fig. 2. Scanning electron microscopy observation on somatic embryogenesis ofOncidiumGower Ramsey. (a) Two somatic embryos started to form on the outer layers of callus (bar=
100mm). (b) Two embryos that have much smaller embryonic
cells than the cells of parent callus (bar=125 mm). (c) A
young embryo-derived PLB with a protrusion in the anterior region (bar=125mm). (d) A young embryo-derived PLB with
two sheath leaves (bar=500 mm).
Repeated subculture of such totipotent callus lines on the original medium resulted in continued proliferation without apparent loss in regeneration capacity for 2 years.
Since our experiments were conducted with only one cultivar, the variants on callusing and embryo formation of other cultivars requires further stud-ies. Also, the genotypic and phenotypic fidelity of the process has to be ascertained before develop-ing any practical clondevelop-ing protocols. The strong totipotency of callus also provided an opportunity to obtain embryogenic suspension culture, and molecular breeding of this orchid.
In conclusion, this report is the first to show that the embryogenic calli derived from the seg-ments of roots, stems and leaves of Oncidium are able to form somatic embryos in a hormone-free
Fig. 3. Plant regeneration. (a) Cluster of embryos/PLBs with a protrusion in the anterior region and absorbing hairs in the posterior region (bar=500 mm). (b) Two young
embryo-derived PLBs with sheath leaves (bar=500mm). (c) Several
embryo-derived PLBs with more expanding sheath-leaves (bar=750 mm). (d) Cluster of embryo-derived PLBs
pro-duced first true leave (bar=1.25 mm). (e) A rooted plantlet recovered from embryos. (f) Potted plantlets with pseudob-ulbs.
embryos on a hormone-free 1/2-MS medium, and these embryos developed into healthy plantlets through PLBs. Root tip-derived callus could be subcultured in past 2 years on the same medium, and still retained strong regeneration capacity and produced somatic embryos in the highest fre-quency (93.8%) and average 29.1 embryos per callus (3×3 mm2) on a basal medium
supple-mented with optimum NAA and TDZ
combinations.
1/2 MS medium. The optimum combinations of NAA and TDZ significantly promoted the fre-quency of embryo formation up to 93.8% in root-derived callus. In addition, most of the somatic embryos converted into healthy plantlets with an almost 100% survival rates when transplanted. The optimized procedure required about 12 – 14 weeks from the initiation of callus to the plantlet formation. This reliable and highly efficient method for embryo formation from callus is essen-tial to establish a cell suspension culture protocol for Oncidium. Furthermore, this method also opens up the prospects of using biotechnological approaches for Oncidium improvement.
Acknowledgements
This paper represents a portion of the first author’s dissertation presented to the Faculty of the Research Institute of Horticulture of the Na-tional Taiwan University in partial fulfillment of the requirements for the Ph.D degree. Experiments were conducted at the Institute of Botany, Academia Sinica at Taipei, Taiwan, Republic of China. The financial support of the Academia Sinica and the National Science Council of the Republic of China are gratefully acknowledged. We thank Dr. Fure-chyi Chen for providing donor plants of Oncidium Gower Ramsey.
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