Springer 2005
Plant Cell, Tissue and Organ Culture (2005) 80: 221–228
Research note
A protocol for in vitro germination and sustainable growth of two tropical mistletoes Sylvia L. P. Ang & Jean W. H. Yong* Natural Sciences Academic Group, Nanyang Technological University, 1 Nanyang Walk, Singapore 637616, Singapore (*requests for offprints: Fax: +1-401-8639733; E-mails: jean
[email protected], whjyong@nie. edu.sg) Received 18 December 2003; accepted in revised form 4 May 2004
Key words: coconut water, cytokinin, Dendrophthoe, Marcosolen, ventilated culture vessels
Abstract We report a protocol for in vitro germination and sustainable growth of two tropical mistletoes, Dendrophthoe pentandra and Macrosolen cochinchinensis. Normal mistletoe shoots with haustoria have been produced using Murashige and Skoog (MS) media supplemented with coconut water (15–20% v/v) in ventilated culture vessels. Germination of mistletoes took place on hormone-free basal MS media, and the time needed for germination was much shorter for Macrosolen than for Dendrophthoe. After germination, further development of the mistletoes required a hormonal input from the medium. We found that 20 lM 6-benzylaminopurine (BA) did allow normal development of only Dendrophthoe, while coconut water (15– 20% v/v) gave complete development of both species in ventilated culture vessels. Multiplication of these mistletoes is also possible via the callus stage using half-strength MS media supplemented with BA at various concentrations in ventilated culture vessels. This is probably the first report of a successful culture for the mistletoe genus of Macrosolen. Compared to Macrosolen, the nutritional and hormonal requirements for Dendrophthoe are less demanding, making the latter a useful model for further mistletoe research.
Mistletoes, unlike holoparasites, are not totally dependent on their host because they possess chlorophyll. However, they still require water, minerals and physical support from their host plant. Some mistletoes derive substantial nutrition by connecting to the host’s phloem, but most obtain their nutritional benefits by tapping on the xylem tissues of the host. Xylem-tapping mistletoes were previously classified as ‘hemi-parasites’, because of their need to obtain water and minerals from their hosts. Marshall and Ehleringer (1990) have shown however that the term is somewhat inaccurate because mistletoes could also obtain a sizable portion of their carbon needs directly from the host xylem. Attention has been given to estimate the proportion of heterotrophic carbon gain by mistletoes (e.g., Richter et al., 1995). The idea of an obligate carbon input in xylem-tapping mistletoes was originally suggested by Raven
(1983), given that these mistletoes receive their nitrogen mainly via nitrogen-containing organic compounds from the host xylem sap. Thus, it would be interesting to assess whether mistletoe could germinate and grow on a suitable culture medium (simulating host plant xylem sap) using plant tissue culture techniques. While germination of mistletoes on tissue culture media has been described (Johri and Bajaj, 1964; Bhojwani, 1969; Hall et al., 1987; Deeks et al., 1999), there are no reports of sustainable growth of mistletoes on these culture media. Growing mistletoes on tissue culture media is a good way to test the hypothesis of whether mistletoes are truly ‘water and nutrient parasites’ by introducing an exogenous source of carbohydrates in the medium. The long-term goal of this research is to produce a ‘complete’ mistletoe (with a haustorium but without a host plant) from either germinated seeds or callus for physiological
222 studies and in vitro screening for potential chemical control agents. This work can potentially allow mistletoes to be cultured without their host plants on a large scale for secondary metabolite production (e.g. anti-cancer agents). Botanical surveys carried out in the last 10 years have shown that Dendrophthoe pentandra (L.) Miq. and Macrosolen cochinchinensis (Lour.) Tiegh. are the two most common mistletoes found in Singapore. They frequently occur on roadside trees and fruit trees (Yong and Hew, 1995; Foo et al., 2003). Ripe fruits of Dendrophthoe and Macrosolen were collected from selected host trees around Singapore (Foo et al., 2003). Freshly collected fruits were rinsed in distilled water for 2– 5 min. The fruits were surface sterilized by soaking in ethanol for 1–2 min (90% v/v for Dendrophthoe; 75% v/v for Macrosolen), followed by continuous agitation for 15 min in sodium hypochlorite (10% v/v Clorox, Singapore) solution, and rinsed three times with autoclaved distilled water. Removal of the pericarp was carried out under aseptic conditions. The ‘‘seeds’’ (endosperms with enclosed embryos) were 6–8 mm in length. These seeds enveloped within the sticky viscin layer, were placed on a solidified basal medium containing a hormone-free Murashige and Skoog (1962) (MS) medium supplemented with 2% sucrose and 0.5% Phytogel as the gelling agent. The pH of all media was adjusted to 5.8 before autoclaving at 121 C for 20 min. All experimental cultures were grown in a tissue culture room at the following conditions: 26 ± 2 C/22 ± 2 C day/night temperature, 10-h photoperiod at 45–55 lmol m)2 s)1 photosynthetic active radiation on the surface of the containers. The light source consisted of eight cool white fluorescent tubes (18 W daylight tubes, OSRAM, Germany). For the germination experiment, the seeds were placed on the solidified media in sterile plastic Petri dishes (60 mm · 15 mm, Sterilin, UK). Each dish contained one seed sitting on 10 ml of basal medium, and was sealed with one layer of Parafilm. A minimum of eight replicates was set up per treatment. After germination, mistletoe seedlings were transferred aseptically to ventilated culture vessels (Magenta GA7 vessels and polypropylene lids with gas-permeable vents, 10 mm diameter vent, 0.22 lm pore size, Sigma, US) containing basal medium (100 ml) with various additions of BA or coconut water (CW) (Hew et al., 1995b). Non-
ventilated culture vessels referred essentially to the same GA7 vessel polycarbonate bottom with a polypropylene lid (Hew et al., 1995b; Hew and Yong, 1997). For the experiments involving BA supplementation, BA was filter-sterilized with a cellulose acetate syringe filter (0.20 lm, Munktell, Germany), before adding to autoclaved MS medium after cooling to ca. 50 C. Three different BA concentrations (5, 10 and 20 lM for Dendrophthoe; 10, 20 and 40 lM for Macrosolen) were tested (Table 1). A control with no BA addition was also set up. The effect of CW supplement on the development of mistletoe seedlings was also investigated. When indicated, fresh CW (15% v/v for Dendrophthoe; 20% v/v for Macrosolen) was added to the MS medium prior to autoclaving. Observations on the growth and development of mistletoe seedling were made periodically. The various preliminary studies carried out with ventilated, and non-ventilated culture vessels indicated that continuous normal development of mistletoe calli and shoots (beyond the six to 10 leaves stage) was only possible in ventilated culture vessels (Table 1). Early senescence of mistletoe shoots was observed in normal (‘closed’ or unventilated) culture vessels despite repeated subculturing onto fresh media. When the mistletoes grew larger than 5–6 cm, larger glass jars (320 mm height · 100 mm; 170 mm height · 125 mm; Ikea Pte. Ltd., Sweden) were used and the gas-permeability of these glass jars was maintained by sealing the opening with a gaspermeable and autoclavable plastic (Fluorocarbon polymer film, Culture Pack-Rockwool System, Toyobo Pte Ltd., Japan; Tanaka 1991). Periodic sub-culturing was carried out once every two to three months. Callus tissues were excised from the shoothaustorium junction of 3-month-old Dendrophthoe or Macrosolen seedlings. The calli were cultured in Petri dishes containing 10 ml half-strength MS media with BA. The Petri dishes were sealed with one layer of Parafilm. It was noted that mistletoe calli did not grow well beyond 3 weeks in Petri dishes due to tissue browning. Hence after 14 days of culture in Petri dishes, the calli were transferred to ventilated GA7 culture vessels containing halfstrength MS media (100 ml) with BA (20 lM of BA for Dendrophthoe and 40 lM of BA for Macrosolen) for further growth and observation (Table 1). Various preliminary studies indicated
223 Table 1. A summary of the different medium and cultural conditions used for the various growth phases of the tropical mistletoes Dendrophthoe pentandra (L.) Miq. and Macrosolen cochinchinensis (Lour.) Tiegh Dendrophthoe Mistletoe growth phase Seed culture Seed germination Seedling shoot emergenceb Early seedling growth Continual growth of mistletoe Callus culture Callus growth with some shoot bud formationd Shoot regeneration
Macrosolen
MS mediaa, Petri dish n.a.c
MS media, Petri dish MS media with either 0, 10, 20, 40 lM BA, or 20% (v/v) CW, ventilated GA7 vessels MS media with either 0, 5, 10, 20 lM BA, or MS media with either 0, 10, 20, 40 lM BA, or 15% (v/v) CW, ventilated GA7 vessels 20% (v/v) CW, ventilated GA7 vessels MS media with either 15% (v/v) CW or 20 lM MS media with 20% (v/v) CW, ventilated glass BA, ventilated glass jar jar 1/2 MS media with 20 lM BA, ventilated GA7 vessels MS media with either 15% (v/v) CW or 20 lM BA, ventilated GA7 vessels
1/2 MS media with 40 lM BA, ventilated GA7 vessels MS media with 20% (v/v) CW, ventilated GA7 vessels
a
Hormone-free MS medium supplemented with 2% sucrose and 0.5% Phytogel as the gelling agent. The stage of shoot emergence (after haustorium establishment) in Macrosolen is shown in Figure 2B. c n.a. = not applicable. d Callus cultures can also be kept in petri dishes but not beyond 2 weeks. b
that half-strength MS media with BA supplementation (20 lM of BA for Dendrophthoe and 40 lM of BA for Macrosolen) gave good shoot bud formation from callus tissues. After 3 months of growth, some of these regenerated shoots were excised and transferred to fresh media (MS media with either 20 lM BA or 15% v/v CW for Dendrophthoe, or MS media supplemented with 20% v/v CW for Macrosolen) for further sustainable growth in ventilated culture vessels (ongoing experiments; mistletoe shoots are normal and growing well after 1 month). After 28 days of culture on basal MS media, about 85% of Dendrophthoe seeds started producing their first leaves (Figure 1A). Following which, these Dendrophthoe seedlings were transferred aseptically to ventilated culture vessels containing the basal medium. In some vessels, the basal medium was supplemented with various concentrations of BA or coconut water (CW) (Figure 1B, E and F). In contrast to the germination rate of Dendrophthoe, germination of Macrosolen seeds was rapid and 85% of the seeds produced their haustoria within 2 days in the basal media (Figure 2A). Within 2–10 days, all uncontaminated Macrosolen seedlings were transferred directly to ventilated GA7 culture vessels containing the basal medium (unsupplemented or with vari-
ous additions of either BA or CW) for further growth and observation (Figure 2B and C). When kept on unsupplemented basal media in ventilated culture vessels, the germinated mistletoe seeds failed to undergo normal shoot development despite culturing for more than 40 days for Dendrophthoe and 21 days for Macrosolen seeds. Under such treatment, the leaves eventually turned brown and wilted off. In general, leaf morphology of Dendrophthoe (Figure 1C) grown in ventilated cultures is similar to those grown naturally on host plants in the greenhouse (Figure 1L). All Dendrophthoe plants developed normal leaves and haustoria, and grew well beyond 90 days in the ventilated GA7 vessels (Figure 1I, J) or glass jars (Figure 1D, H) where the basal media was supplemented with either BA or CW. Multiple shoot bud formation from the callused portion was observed at the shoot-haustorium junction when Dendrophthoe was cultured on media supplemented with either 10 or 20 lM of BA (Table 2). In terms of leaf size, Dendrophthoe leaf development was optimized in 20 lM BA treatment (Figure 1J). Dendrophthoe callus formation at the shoot-haustorium junction was stimulated by all three concentrations of BA tested, with 10 lM BA producing the most calli (Table 2).
224
Figure 1. In vitro germination and growth of the tropical mistletoe Dendrophthoe pentandra (L.) Miq. (A) The first pair of leaves emerged after 28 days in culture containing basal MS medium. (B, C) Young Dendrophthoe after 50 and 130 days, respectively, in a ventilated GA7 vessel containing MS medium supplemented with 15% v/v coconut water. (D) Dendrophthoe after 9 months in a ventilated glass jar containing MS medium supplemented with 15% v/v coconut water. (E, F) Young Dendrophthoe after 50 days in a ventilated culture vessel containing MS medium supplemented with 5 and 20 lM BA, respectively. (G) Dendrophthoe calli in a ventilated GA7 vessel containing half-strength MS medium supplemented with 20 lM BA. (H) Dendrophthoe after 1 year in a tall ventilated glass jar containing MS medium supplemented with 15% v/v coconut water. (I, J) Dendrophthoe after 90 days in a ventilated GA7 vessel containing MS medium supplemented with 5 and 20 lM BA, respectively. (K) Regeneration of new Dendrophthoe shoots from calli after 60 days in a ventilated culture vessel containing half-strength MS medium supplemented with 20 lM BA. (L) Young Dendrophthoe growing naturally on a host branch in a greenhouse in Singapore. Note: There were at least 8 replicates per treatment. Representative mistletoe is shown here.
225
Figure 2. In vitro germination and growth of the tropical mistletoe Macrosolen cochinchinensis (Lour.) Tiegh. (A) Germination and growth of the haustorium tip after 10 days on basal MS medium. (B) The first pair of leaves emerged after 20 days (after the haustorium has been formed). MS medium supplemented with 20 lM BA. (C) Straightening of the young shoot and elongation of the first pair of leaves at 40 days. MS medium supplemented with 20 lM BA. (D) Young Macrosolen growing naturally on a host leaf in a greenhouse in Singapore. (E, F) Macrosolen after growing for 110 and 140 days, respectively, in a ventilated GA7 vessel containing MS medium supplemented with 20% v/v coconut water. (G) Macrosolen after 260 days in a ventilated glass jar containing MS medium supplemented with 20% v/v coconut water. (H, I, J) Young Macrosolen after 48 days in a ventilated culture vessel containing MS medium supplemented with 10, 20 and 40 lM BA, respectively. (K) Regeneration of Macrosolen shoots from calli after 3–4 weeks in a ventilated culture vessel containing half-strength MS medium supplemented with 40 lM BA. Note: There were at least 8 replicates per treatment. Representative mistletoe is shown here.
Leaf morphology of Macrosolen (Figure 2E, F) grown in ventilated cultures is similar to those grown naturally on host plants in the greenhouse (Figure 2D). The presence of BA in the MS media assisted the process of Macrosolen shoot straight-
ening and leaf emergence (Figure 2B, C), with 20 lM BA giving the best results after 35 days (Table 2). Some Macrosolen shoot bud formation from the callused portion was observed at the shoot-haustorium junction when cultured on MS
226 Table 2. Effect of 6-benzyladenine supplementation on the growth of tropical mistletoes Dendrophthoe pentandra (L.) Miq. and Macrosolen cochinchinensis (Lour.) Tiegh. on MS media in ventilated GA7 vessels BA (lM)
Dendrophthoe Callus growth indexa
0 5 10 20 40
++ ++++ +++ n.a.d
Macrosolen Average leaf number after 84 days (Mean ± S.E.b)
Callus growth indexa
Average leaf number after 90 days (Mean ± S.E.b)
None survived 7.0 ± 1.1 7.0 ± 1.6 6.6 ± 1.2 n.a.
n.a.d ++ + +
None survived n.a. 4.5 ± 0.3 12.5 ± 2.5 4.6 ± 0.6
Percentage of plants with fully emerged seed leaves after 35 daysc
n.a. 67 100 67
Callus growth, at the shoot-haustorium junction, was rated by the index as follows : ), no callus; +, poor; ++, fair; +++, good; ++++, very good. b S.E. = standard error. Note: There were at least 8 replicates per treatment. c The stage of shoot emergence (after haustorium establishment) in Macrosolen is shown in Figure 2B. d n.a. = not applicable. a
medium supplemented with BA (Figure 2H–J). The addition of 20 lM BA in the medium increased the number of leaves produced (Figure 2I; Table 2). In contrast to Dendrophthoe where a MS medium with 20 lM BA supplementation is sufficient to promote normal development, Macrosolen could only grow beyond 260 days with normal shoots and haustoria in media supplemented with CW (Figure 2G). As observed earlier for the young mistletoes, callus tissues obtained from Dendrophthoe were also prone to browning when kept in ‘closed’ culture vessels (non-ventilated) beyond 3 weeks. Excised calli (Figure 1G) from Dendrophthoe grew well on half-strength MS supplemented with 20 lM BA in ventilated vessels. The shoot buds (Figure 1K) on the callused portion were able to develop into a complete shoot later by sub-culturing the excised shoots in ventilated culture vessels containing MS media with either 20 lM BA or 15% v/v CW (ongoing experiment). This shows that multiplication of Dendrophthoe is possible via the callus stage. The excised calli from Macrosolen also grew well in half-strength MS supplemented with 40 lM BA. The callused portion produced tiny shoot buds after 3–4 weeks in culture (Figure 2K). As observed for Dendrophthoe, these tiny shoot buds could develop into a normal shoot subsequently by sub-culturing them on MS media supplemented with 20% v/v CW in ventilated cultures (ongoing experiment). Hence
multiplication of Macrosolen is also possible via the callus stage. As reported previously (Bhojwani, 1969; Hall et al., 1987; Deeks et al., 1999), mistletoes did germinate on tissue culture media and this indicates that mistletoe germination is probably not host-specific, at least for the two tropical species tested in this paper. After germination, further development of the mistletoes requires a hormonal input from the medium. In a natural setting, the mistletoe will receive some hormonal inputs (e.g., cytokinins) from the host xylem sap through its haustorium. Xylem sap contains various hormones such as cytokinins (e.g., Yong et al., 2000), in addition to water and mineral nutrients. In this study, we found that BA (an aromatic cytokinin) supplementation on MS media did enable normal shoot development of Dendrophthoe. Furthermore, adding CW to the MS media stimulates complete development for the two species tested. CW (10–20% v/v) has been found to be beneficial for a wide range of morphogenetic manifestations in plant tissue cultures (for a review, see George, 1993). It is suggested that the addition of CW serves to rejuvenate mature cells and promote cell division through cytokinins. CW is considered to be a crude and rich source of cytokinins (e.g., Kobayashi et al., 1995). In contrast to CW, a fully defined synthetic medium will not have all the components required for growth, especially the uncharacterized plant hormones and
227 organic components (George, 1993). Thus, it is noteworthy that cytokinins are important hormones for growth and development of mistletoes, particularly Dendrophthoe. At present, the role of the other hormones (gibberellic acid, auxins, brassinosteriods, etc.) in regulating mistletoe development remains unclear and cannot be ruled out. It is well established that plant growth in conventional closed systems may be inhibited by changes in the gas composition, particularly water vapour, ethylene, and CO2 (for a review, see Buddendorf-Joosten and Woltering, 1994). Most plant parts produce ethylene gas and high levels of headspace ethylene are unfavourable for plant growth because it promotes senescence (Buddendorf-Joosten and Woltering, 1994; Hew et al., 1995a, b). In a conventional closed culture system, water vapour and ethylene gas accumulate as a result of the restriction of gas exchange between the culture vessel and the external environment. In our study, early senescence of mistletoe shoots and calli has been observed in normal (unventilated) culture vessels. Such problems can be alleviated by the use of ventilated GA7 vessels or glass jars. With better ventilation, water relations of the shoots could be improved and the venting of headspace ethylene could be more efficient. These factors are probably responsible for the improved growth of mistletoes in ventilated cultures. Clearly more work needs to be done in identifying the main factor(s) responsible for promoting better growth of mistletoes, and also of other plants in ventilated cultures. In conclusion, normal mistletoe shoots with haustoria have been produced using MS media supplemented with CW in ventilated culture vessels. This is the first report of a successful culture for the mistletoe genus of Macrosolen. MS media supplemented with BA could allow normal development of Dendrophthoe but not of Macrosolen. Multiplication of the tropical mistletoes is possible via the callus stage using half-strength MS media supplemented with BA in ventilated culture vessels. There is room for further optimization of this protocol, and for evaluation on the other mistletoe genera of Amylotheca, Helixanthera, Scurrula and Viscum. Compared to Macrosolen, the nutritional and hormonal requirements for Dendrophthoe are less demanding, making the latter a useful model for further mistletoe research. This research, using
plant tissue culture techniques, has teased apart the obligate and parasitic association between mistletoe and its host, and paved the way for further research and applications involving mistletoes without their host plants.
Acknowledgements We thank Prof Stuart Letham and Prof Michio Tanaka for their invaluable discussion, and generous support of culture vessels and gas-permeable plastics. The technical support given to this project by the Ngee Ann Polytechnic students (Mr Kim Chen Sze, Mr Seet Thong Sen and Mr Teh Khai Ping) is acknowledged. The suggestions given by the anonymous reviewer are much appreciated. We are grateful to the National Parks Board (Singapore) for granting us permission to collect the mistletoes. This work was supported by a grant (RP YWH 01/14) from the National Institute of Education, Nanyang Technological University, Singapore.
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