The Impact of Environmental and Host Specificity in Seed Germination and Survival of Korean Mistletoe [Viscum album var. coloratum (Kom.) Ohwi]

Abstract

Humankind has been searching for medicinal materials from various plant sources in an attempt to treat disease. Mistletoe is one indubitable plant source for these materials due to its effectiveness in treating various diseases, but it has almost disappeared from the mountainous areas of Korea due to excessive harvesting. In this study, in order to select host tree species for Korean mistletoe [Viscum album var. coloratum (Kom.) Ohwi] by seed inoculation and to clarify the effect of host specificity among various tree species were conducted for the purpose of gaining basic information for the artificial cultivation of Korean mistletoe. Almost all the seeds of Korean mistletoe germinated in vitro at the temperature of 15℃. Among host trees used in this study, Prunus mume showed the highest parasitic affinity with inoculated Korean mistletoe, compared with any other host plants. However, treatment of hormones could not increase the low survival rate of Korean mistletoe on the host trees.

Introduction

As an evergreen parasitic plant, Viscum album forms adventitious roots to absorb water and nutrients from its host plant for photosynthesis (Visser, 1981). In Korea, V. album has a hemiparasitic relationship with Quercus spp. (e.g. Quercus dentata), Celtis sinensis, Betula platyphylla var. japonica, Salix koreensis, Alnus japonica, and Castanea crenata var. dulcis (Kim, 1996; Lee, 1985; Ministry of Agriculture, 2000; Ministry of Agriculture, 2007). Interestingly, Lee (2009) reported that Korean mistletoe [Viscum album var. coloratum (Kom.) Ohwi] was observed on both autogenously grown conifers including Pinus densiflora and Pinus koraiensis, and on naturally grown on broad-leaved trees, such as Zelkova serrata, Diospyros kaki, Acer mono, Acer palmatum, Morus alba, Kalopanax pictus, Ginkgo biloba, in the regions where a colony of mistletoe was observed. It is widely known that host specificity of Korean mistletoe have a lot of species of trees. According to Hariri et al. (1991), however, host plant may have certain defense mechanisms that will be generating during invasion of endophyte. In forming a physical connection between the mistletoe and host organization, it is suspected that presence of the chemical defense mechanism. This result suggests that Korean mistletoe may have host specificity.

V. album has long been used as a medicine for various diseases; lectins and viscotoxins found in the plant are well-known as biologically active proteins and polypeptides (Machaidze et al., 1996; Tubeuf, 1923). Recently, there has been increasing attention to its efficacy in chemotherapy; mistletoe extracts are being used as a new anti-cancer substance. Anti-cancer activity of the mistletoe has been reported due to the cytotoxic and immunomodulating substances (Büssing, 1999, 2000). In Korea, many studies for metabolite identification and pharmacological actions of mistletoe extracts have been conducted (Kim, 1997; Park, 1999; Sung, 2005; Yoon, 1997; Lee, 2002; Lee, 2003; Jang, 1997; Jun, 2004; Jung, 1999; Jung, 2005; Choi, 2005). By comparing Korean and European mistletoe, it was discovered that the two species have different lectin components, which cause different antibody responses (Lyu et al., 2000). Choi (2005) reported that DNA sequence alignments between the Korean and European mistletoe did not correspond to each other. Lee (2003) also demonstrated, through research measuring physiological activities from four different varieties, that metabolite compositions in mistletoe extract depend on the host plant.

Plant growth regulators are adjustable to a particular growth response in plant; mainly it represents the plant hormone. The plant hormone auxin and cytokinine promote the plant growth and differentiation of cell. It has been known that control of development of shooting and rooting is depending on the rate of auxin and cytokinine. According to the experiment of Martin et al. (2012) that treated various concentrations of IAA and kinetin to mistletoe, health of mistletoe explant was related to the concentration of IAA. At an IAA/kinetin ratio of 0.1, also, mistletoe explant was healthiest.

As mistletoe efficacy has been demonstrated in the medical field, and the need for dietary supplements has increased, mistletoe has been indiscriminately taken by people picking medical herbs so that autogenously grown mistletoe cannot be found easily, even in some national parks. Therefore, there is a strong demand for artificial cultivation of mistletoe due to the depletion of natural mistletoe. In order to accumulate fundamental information for the development of artificial mistletoe propagation, we conducted inoculation experiments of mistletoe seeds to investigate host affinity and to verify the parasitic mechanism.

 

Materials and Methods

Seed collection and germination of Korean mistletoe

To collect Korean mistletoe seeds, naturally grown 25-years-old Viscum album var. coloratum (Kom.) Ohwi seeds were taken from an approximately 35-years-old Quercus variabilis host tree growing on Mt. Jiri (Sannae-myeon, Namwon). To check the result of Lamont (1983) that 15℃ was the optimal temperature for mistletoe seed germination, comparative germination tests were carried out with the ambient temperature. 25 mistletoe seeds were placed on filter paper wetted with distilled water in each of the two petri dishes with a cover and incubated to keep relative humidity at 70% in chamber setting 15 and 25℃. This test designated as one replication and the test was performed five times. After three weeks, seeds with hypocotyls that were 1 mm or longer were counted as germinated seeds.

Host specificity of Korean mistletoe seed

Based on the previous report on mistletoe habitats conducted by Lee (2009), Quercus variabilis, Castanea crenata var. dulcis, Prunus serrulata var. spontanea, Prunus mume, and Malus pumila var. dulcissima were selected as potential candidates to determine host affinity with artificially inoculated mistletoe seeds. Mistletoe seeds were taken from an approximately 20-year-old Viscum album var. coloratum (Kom.) Ohwi parasitizing on a Quercus variabilis tree on Mt. Jiri (Sannaemyeon, Namwon) on February 28, 2005. Before this test, seeds were stored at −2℃ for 15 days. For seed inoculation, the mistletoe pericarp was manually removed and 10 peeled seeds were then inoculated at 10- cm intervals, via the adhesiveness of their internal flesh, onto the two-year-old branches of the five host plants. The inoculated branches of each host were designated as one replication and the test was performed thrice. This inoculation was conducted in the experimental field installed in Susanri area (Sudong-myeon, Namyangju). In the spring of the next year after inoculation, the degrees of affinity were calculated by investigating whether cotyledon grew from the parasitic seeds.

Field test for mistletoe seed inoculation

From the five species in the host specificity test, Prunus mume was selected as a host tree for the field test of seed inoculation. Also, Morus alba was selected as a control their medium-sized heights are suitable for relatively easy management. Two-meter-tall P. mume and M. alba, grown for three years after their propagation, were transplanted in experimental city (Sudong-myeon, Namyangju) for this seed inoculation test. Seeds were collected from an approximately 20-year-old Viscum album var. coloratum (Kom.) Ohwi parasitizing on a Quercus variabilis tree on Mt. Jiri (Sannaemyeon, Namwon) at the end of February 2006, and stored at −2℃ for 15 days. The mistletoe pericarp was manually removed and 5 peeled seeds were then inoculated at 10- cm intervals, via the adhesiveness of their internal flesh, onto the two-year-old branches of the host plants. The inoculated branches of each host were designated as one replication and the test was performed five times. After the first year following inoculation, seed germination on the bark of host plants and hypocotyl elongation were investigated. The survival rates of mistletoe seeds were investigated during the spring of the second year. At the following fall, survival rates of germinated seeds were finally judged. Three years after inoculation, the numbers of mistletoe branches were counted.

Effects of hormone treatment for survival

To investigate the effect of hormones on the survival of hypocotyl, plant hormones treated in germinated mistletoe hypocotyls. M. alba was selected as the host in this hormone treatment test because inoculated mistletoe seeds on this tree showed low survival rates in the preceding test for screening host specificity of mistletoe. Seeds were taken from an approximately 20-year-old Viscum album var. coloratum (Kom.) Ohwi parasitizing on a Quercus variabilis tree on Mt. Jiri (Sannae-myeon, Namwon), at the end of February 2007 and stored at −2℃ for one month. After germination and hypocotyl elongation of inoculated mistletoe was complete, either 100 ppm or 200 ppm of IAA or kinetin hormones were applied between the mistletoe haustorium and the host bark used a brush.

 

Results

Seed germination of Korean mistletoe

To verify whether mistletoe seed germination is directly affected by temperature, an in vitro seed germination experiment was conducted. As shown in Fig. 1, seed germination rates of incubated mistletoe could be determined by observing hypocotyl emergence after about 20 days, and these hypocotyls grew up to 5 mm over time (Fig. 1. B). The germination rate of mistletoe seeds at 15℃ was 92.8%, while the germination rate at 25℃ was 63.2%, it was shown a significant difference (Table 1; p < 0.001, F = 18.50).

Fig. 1.Germination of Korean mistletoe seeds: seeds arranged on the petri dish (A) and hypocotyl elongated from seed (B).

Table 1.Germination rate in vitro under the different temperatures

Host specificity of Korean mistletoe seed

From the test to investigate the host-parasitic compatibility of Korean mistletoe with three species of Rosaceae and two species of Fagaceae, it was determined that seed germination, hypocotyl elongation, and haustorium plate formation of mistletoe were viable after one year on the branches of all of the tree species tested. As time went on, almost all seeds of inoculated mistletoe germinated and elongated their hypocotyls, and any differences caused by the five host species could not be detected. During the formation of mistletoe haustorium, any difference among the five trees was not shown. Regardless of the surface conditions of the host, mistletoe developmental steps from germination to haustorium formation successively progressed. As suggested by Tubeuf (1923), host plants might not provide mistletoe with any developmental influence during these periods. It was observed that new leaf emergence from the mistletoe hypocotyls at the spring of the second year was rare, as low as several percent, except on P. mume (Fig. 2). By the way, almost all the hypocotyls on the branches of P. mume survived to the autumn of the second year and elongated new leaves on their top at the spring of the third year, showing that the compatibility of Korean mistletoe with P. mume is surprisingly high, compared with the other tree species tested in this experiment (Fig. 4).

Fig. 2.Changes in hypocotyl survival on the host plant species at the next spring after inoculation of Korean mistletoe seeds. Vertical bars indicate standard error of the means (n = 3).

Fig. 4.Growing process of Korean mistletoe on the host P. mume after inoculation of seeds: seed germinated and elongated hypocotyl in 1st spring (A) and autumn (B); first leaves on the top of the hypocotyl and a new stem near the hypocotyl emerged in the 2nd spring (C) and autumn (D); a second set of leaves grew from a new node, which emerged on the first stem in the 3rd spring (E) and autumn (F); all stems grew vigorously in 4th spring (G) and autumn (H).

Field test for mistletoe seed inoculation

Table 2 describes the rates of inoculated seed germination, hypocotyl elongation, adhesive disk formation, and survival. Inoculated seed germination and adhesive disk formation were excellent on both P. mume and M. alba, and any difference between the two species was not detected. These results are consistent with the results of Tubeuf (1923), who suggested that the host surfaces in direct contact with mistletoe seeds did not affect seed development. Moreover, this result implies that mistletoe does not have any specificity until hypocotyl elongation and disk formation, in addition to seed germination. While the survival rate of mistletoe seed inoculated on P. mume in the second year of inoculation was great (p < 0.001, F = 69.28), mistletoe seeds inoculated on M. alba showed extremely low survival rates, as low as 2.4%, and all of the surviving seeds were dead the following autumn. From these results, mistletoe is parasitic against P. mume, but does not have a parasitic relationship with M. alba.

Table 2.Comparison of germination, hypocotyl elongation, adhesive disk formation, and stem survival between P. mume and M. alba

Fig. 3 shows an increase in the number of nodes after artificial inoculation of mistletoe seeds for four years. The numbers of stems at the first year represent the number of newly growing hypocotyls, and those at subsequent years represent the number of nodes derived from newly grown hypocotyls with more than two leaves. While most mistletoe seeds inoculated on M. alba were dead in the autumn of the second year, as shown Table 2, on P. mume one to two (rarely three) hypocotyls developed from a single seed, and these hypocotyls started to penetrate the hosts’ bark by forming endophytes. Also, the total number of stems increased, depending on the stems newly formed by the endophyte. These results are consistent with the schematic of the endophyte system suggested by Heide-Jørgensen (1989). Once again, after the third year, further increases in the number of stems were observed, and at the fourth year, the number of mistletoe stems that developed from one seed was more than two and a half.

Fig. 3.Increase in the number of stems (hypocotyls at year 1) after the inoculation of Korean mistletoe seeds. Vertical bars indicate standard error of the means (n = 5).

Figures 4 and 5 shows the spring and autumn development of mistletoe seeds inoculated on P. mume and M. alba, respectively. For the first year, there were no differences between the inoculated seeds on the two plants investigated. Beginning in the second year, the number of mistletoe stems gradually declined and the inoculated seeds showed a 2.4% survival rate on M. alba. At the third year, the remaining mistletoe on M. alba was completely dead over the summer, whereas mistletoe on P. mume was vigorously growing.

Fig. 5.Growing process of Korean mistletoe on the host M. alba after inoculation of seeds: seed germinated and elongated hypocotyl in 1st spring (A) and autumn (B); the first leaves on the top of hypocotyl emerged, but lost vigor in the 2nd spring (C) and autumn (D); first stem stopped growing in the 3rd spring (E) and finally died in the 3rd autumn (F)

Effects of hormone treatment for survival

To increase the survival rate of mistletoe, IAA and kinetin hormone were used to treat the inoculated seeds. Among the plant hormones, auxin and cytokine have been known to promote plant cell division, so we had expected that both hormones might have an effective impact on the formation of endophyte. In the spring of the year following mistletoe seed inoculation on M. alba, the survival rate was calculated by counting the newly development leaves of mistletoe seeds. Compared to the control without hormone treatment, however, treatments of either IAA or kinetin hormone did not increase the survival rate (Fig. 6).

Fig. 6.Changes in hypocotyl survival rate after the inoculation of Korean mistletoe seeds on the host M. alba, according to the treatment of IAA and Kinetin hormones. Vertical bars indicate standard error of the means (n = 5).

 

Discussion

In the seed germination experiment designed for testing whether mistletoe germination is controlled by temperature at the beginning stage of seed germination, almost all of the seeds from Korean mistletoe grown on the Quercus variabilis germinated in vitro at the temperature of 15℃. However, the rate of seed germination at the temperature of 25℃ was 63 percentage. This result coincides with the suggestion by Lamont (1983) and suggests that seed germination is affected by temperature. In the testing of effect on the germination by the host plants, however, the initial germination rate was excellent, no matter the species. This suggests that seed germination is not affected by the host species and is not the limiting factor for the early steps of artificial propagation. It is reported by Stopp (1961) that European mistletoe has dormancy and light affects mistletoe seed germination. Thus, scrupulous research on the relationship between dormancy and light is required. In addition, the collecting period and method for artificial cultivation of mistletoe have to be considered.

In the test for host specificity of mistletoe seed, in April of the second year after inoculation, the survival rates of inoculated mistletoe seeds were extremely low on all host species except on P. mume. This result is consistent with the artificial propagation experiment of mistletoe conducted by the Ministry of Agriculture (2000), which demonstrated that inoculated mistletoe seeds on all host species used in the tests showed extremely low survival rates of less than five percent. The survival rate of inoculated seeds was low on M. pumila. Surprisingly, the fact that mistletoe seeds inoculated on P. mume have very high survival rates is an exciting discovery; further in-depth study on the host specificity of Korean mistletoe is required.

Another trial to artificially cultivate Korean mistletoe by inoculation of seeds on P. mume and M. alba showed an extremely low survival rate on the branches of M. alba in the second year after seed inoculation and death after poor growth in the third year. On the other hand, there was a very high survival rate and continuously vigorous growth of mistletoe on the branches of P. mume, suggesting the possibility of artificial cultivation of Korean mistletoe with the selection of proper host species. In this new method, P. mume can be regarded as one of the best host plants for the artificial cultivation of mistletoe because it showed high parasitic affinity with Korean mistletoe.

In order to increase the survival rate on M. alba, inoculated mistletoe was treated with IAA and kinetin hormone. The treatments of IAA and kinetin at the stage of haustorium development could not reverse the low survival rate of Korean mistletoe on M. alba. It is possible that these hormones could not influence haustorium development and endophyte formation, or that the physical defense mechanisms of the host plant, M. alba, prevented endophyte invasion by parasitic mistletoe. This is supported by another study, which demonstrated that the treatment of IAA and kinetin has no effect on the seed germination of European mistletoe (Luther and Backer, 1986). These results suggest that mistletoe cell division and development at the period of germination or haustorium formation are not significantly affected by the treatment of exogenous auxins and cytokinins.

In this study, we suggest the possibility for artificial propagation of Korean mistletoe at a practical level, which may significantly contribute to the stable supply of materials for medicines and the conservation of natural resources.