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ISSN : 1598-5504(Print)
ISSN : 2383-8272(Online)
Journal of Agriculture & Life Science Vol.54 No.4 pp.37-42

The Optimal In Vitro Culture Condition for Shoot Multiplication of Evergreen Oak Tree, Quercus myrsinaefolia Blume

Dong-Jin Park1, Seong-Hyeon Yong2, Myung-Suk Choi2,3*
1Department of Seed and Seedling Management, National Forest Seed and Variety Center, Chungju, the republic of Korea, 27495
2Division of Environmental Forest Science, Gyeongsang National University, Jinju, the republic of Korea, 52828
3Institute of agriculture and Life Science, Gyeongsang National University, Jinju, the republic of Korea, 52828
*Corresponding author: Myung-Suk Choi Tel:
+82-55-772-1856 E-mail:
July 8, 2020 July 20, 2020 July 21, 2020


The evergreen oak tree, Quercus myrsinaefolia Blume, is not only economically important for wood, medicine, landscape trees, etc., but also becoming more important in terms of ecology due to climate change. However, asexual reproduction was difficult, so this study was conducted to establish the optimum conditions for micropropagation by shoot multiplication. The surface sterilized seeds of Q. myrsinaefolia were successfully germinated in WPM basal medium. BAP (1.0 mg/L) treatment was most effective for inducing multiple shoots. The highest induction rates of adventitious roots from the multiple shoots was shown in the treatment of 1.0 mg/L NAA. Both MS and WPM medium were most effective for growth of multiplied plantlets. For ex vitro acclimatization, the survival rates of multiplied plantlets were 100% in vermiculite and commercial soil. The results of this study can be used for proliferation and supply, and establishment of ex situ conservation of Q. myrsinaefolia elite trees.


    Korea Forest Service


    Quercus myrsinaefolia is one of evergreen broadleaf trees which are natively grown in Korea peninsula. Five species of evergreen Quercus arbor, including Q. myrsinaefolia, are mainly distributed in Jeju Island and southern shore area of Korea. They are considered economically important tree species for timber, medicine, land scape and food. Recently, their importance is increased in the aspects of not only economy but also ecology due to climate change.

    It is predicted that their natural habitats shift to the north and higher altitude according to a climate change scenario in Korea peninsula (National Institute of Forest Science, 2015). The evergreen Quercus trees have been listed in the recommended afforestation species for preparing climate change. Physiological evidences which the evergreen Quercus arbors are tolerant to heat stress and drought-heat dual stress (Park, 2016). Among five evergreen Quercus arbors, Q. myrsinaefolia was most tolerant to cold stress (Park et al., 2019). Therefore, it is expected that demand on this tree species in the market is increased.

    An efficient asexual propagation method for Q. myrsinaefolia was established in this study. Because of their economical and ecological value, asexual propagation of Quercus species through in vitro culture methods have been reported on various species: Q. robur (Favre and Juncker, 1987;San-Jose et al., 1990), Q. petraea (Chalupa, 1993), Q. suber (Manzanera and Pardos, 1990), and Q. alba and Q. rubra (Schwarz and Schlarbaum, 1993;Vengadesan and Pijut, 2009). However, evergreen Quercus species were poorly reported in previous studies on asexual propagations. The only report on asexual propagation method of Q. myrsinaefolia by stem cuttings has been reported by McGuigan et al., (1996). According to this report, there are many limitations, such as quality of materials and time for practicing, for successful propagation using stem cutting. Furthermore, it has been reported that root induction rates were very low on the propagation of Q. acuta by stem cuttings (Kang et al., 2017). Major afforestation species, such as scot pine, poplar, and eucalyptus, have been successfully multiplied using in vitro culture technique (Diego et al., 2010;Clare & Gerry, 2006;Ma et al., 2011).

    In vitro culture has been used as one of most effective ways for plant asexual proliferation. In vitro multiplication of these plants can provide a more efficient alternative to traditional multiplication methods. (Buendía-González et al., 2007). In general, it is well known that in vitro propagation methods of plants are greatly influenced by internal and external conditions such as light, temperature, pH and composition of the medium. Among other things, the composition of the culture medium is very important for the growth of shoots, and the type of medium used for tissue culture depends on the type of plant used and the purpose of culture (Barlass & Skene, 1978;Harris & Stevenson, 1982). Although in vitro propagation is known to be very good for asexual growth of Quercus spp., no research has been conducted on Q. myrsinaefolia. Therefore, this study was conducted to establish optimal conditions for in vitro propagation of Q. myrsinaefolia, a useful plant resource.

    Materials and Methods

    1. Plant materials and in vitro cultures

    In order to establish optimal conditions for in vitro propagation of Q. myrsinaefolia, seeds of superior individuals were germinated. The seeds were collected from the experimental forest in Gyeongsang National University, Jinju, in September. The surface sterilization was performed by washing seeds with 70% EtOH for 2 minute and 4% NaClO for 1 minute (5 times). The seed was inoculated in the vessels containing MS(Murashige and Skoog, 1962) basal medium with 3% sucrose. Culture medium was adjusted pH5.7 and solidified by 0.4% gelrite for germination. Subculture was performed in the same medium every 4 weeks. In the experiment, plants used after growth over 10 cm length for 1 or 2 months.

    2. Shoot multiplication

    The 5 cm in vitro stem was cut and cultured in MS medium containing various growth regulators. For multiple shoot induction, 4 kinds of cytokinin (1.0 mg/l BAP, 1.0 mg/l 2iP, 1.0 mg/l Kinetin and 1.0 mg/l TDZ) were treated by being included in MS basal media.

    3. Elongation of multiplied shoots

    For screening optimal growth media, 5 kinds media; MS, B5(Gamborg et al., 1968), SH (Schenk & Hildebrandt, 1972), QL (Quoirin & Lepoivre, 1977), and WPM (McCown and Lloyd, 1981) were used. In vitro multiplied shoots were collected and transferred to MS basal medium supplemented with 3% (w/v) sucrose. The shoot elongation was carried out by culturing each shoot on 5 kinds of culture media containing 3% (w/v) sucrose without growth regulators. The experimental data was collected after 4 and 8 weeks of culturing

    4. In vitro root induction

    For root induction, 3 kinds of auxins (1.0 mg/l NAA, 1.0 mg/l IAA and 1.0 mg/l IBA) were treated by being included in MS basal media. These tests were carried out at 25±1°C with a photoperiod of 16 hrs of light with 37 μmol m-2s-1 (about 3,000 lux)/8 hrs of the dark for 4 weeks.

    5. Acclimatization of propagated plantlets

    To test the most effective soil for hardening in vitro cultured plantlets, 4 types of soils (Vermiculite, Peatmoss, Pearlite and commercial soil) were used. In vitro cultured plantlets were transferred to each type of soils and incubated in the plant growth chamber (SH-301, Seyeong Science Co., the Republic of Korea) for 4 weeks. The humidity in the plant growth chamber was reduced gently during incubation.

    6. Statistical analysis

    Data are expressed as an average of five separate experiments. The bars indicate standard deviation from the mean of each replicate treatment. Data were subjected to statistical analysis by using the SPSS software. One-way analysis of variance (ANOVA) was conducted, and means were compared using Duncan’s multiple- range test (DMRT) at 0.05 level of probability.


    1. Shoot multiplication

    To establish efficient multiple shoot induction, four kinds of cytokinins were treated: BAP, 2iP, Kinetin and TDZ (Fig. 1 and Table 1). The most effective cytokinin was BAP for induction of multiple shoots. When BAP was treated, the number of the induced primary stem was 2.2±0.2, and the number of secondary stems was 0.6±0.1. 2iP treatment, also, induced a higher number of multiple shoots (primary stems: 2.0±0.3; secondary stems: 1.2±0.5) than the number of multiple-shoot induced by no cytokinin treatment. However, treatments of kinetin or TDZ were less effective than no cytokinin treatment.

    2. Elongation of multiplied shoots

    For efficient in vitro culture conditions, the optimal culture medium for shoot growth of Q. myrsinaefolia was determined. The culture media used in this study were MS, B5, SH, QL and WPM. (Fig. 2). In vitro cultured plantlets were inoculated into each media and cultured for 4-8 weeks. The highest shoot growths were obtained in the MS (1.47±0.15 cm), and WPM (1.43±0.20 cm) media. In the B5 medium, only 0.13±0.03 cm of the shoot was grown, and it was worst effective on shoot growth. Using QL and SH media, 0.73±0.88 cm and 0.53±0.188 cm of shoot growths were obtained.

    3. In vitro root induction

    To establish efficient root induction, three different of auxin were treated: NAA, IAA and IBA (Fig. 3). The most effective auxin was NAA for root induction. The root induction rate was 77.8±11.1% when NAA was treated. When IBA and IAA were treated, root induction rates 33.3±0.0% and 22.2±11.1%, respectively. However, the root induction rate of no auxin treatment was 11.1±11.1%.

    4. Acclimatization of prapagated plantlets

    The most effective soil for hardening in vitro cultured plantlets was screened. Four types of soils were used for acclimatization (Fig. 4). In vitro cultured plantlets were transferred to each type of soils and incubated in the plant growth chamber for 4 weeks. The humidity in the plant growth chamber was reduced gently during incubation. All plantlets transferred to vermiculite and commercial soil were well-taking root and survived. When transfer in vitro cultured plantlets to pearlite and peat-mos, their survival rates were 66.7±14.4% and 41.7±0.1%, respectively. Thus, transferring to vermiculite and/or commercial soil was most effective for hardening in vitro cultured plantlets of Q. myrsinaefolia. Acclimatized plants showed new leaves after 2 weeks and grew normally.


    There have been a variety of asexual propagation studies on Quercus spp., but not on evergreen Quercus spp. It is considered that difficulties low efficiency of practice according to character of the species are major reason for less reports on asexual propagation. This study the first report on in vitro culture condition for asexual propagation of Q. myrsinaefolia was established in this study.

    The shoot multiplication method is very good for plant propagation. Regenerating plants from axillary buds is the most genetically stable method since organs do not de-differentiate and organogenesis occurs immediately (Sanches-Gras & Calvo, 1996). Because of its shortness, it can be said to be the most practical as a means of rapid mass propagation (Bhattacharya & Bhattachraya, 1997).

    Appropriate cytokinin application is important for shoot multiplication. BAP treatment was most effective for multiple shoot induction (Fig. 1). BAP treatment is known to be useful for multiplication of stems. Purohit et al. (2002) also reported that 22.19 μM BAP treatment was most effective in inducing multiple shoots of Q. leucotrichophora. Martinez et al. (2017) also reported that in the cultivation of Quercus ilex L, BAP was effective in multiple shoot proliferation when the culture period was reduced.

    The optimal media for mass propagation of Q. myrsinaefolia were MS and WPM media (Fig. 2). Various media are used for in vitro propagation of woody plants. Most commonly, MS and WPM media are used. Plants generally require different minerals for different species. The medium suitable for the growth of woody plants is not only the presence of these mineral components, but also their concentration and presentation. The main change is generally the nitrate/ammonium ratio as well as the amount of nitrogen affecting the growth of woody plants. Ethylene, a gaseous PGR produced by cultured plant tissue, causes bud growth abnormalities such as bud peak necrosis and leaf resection (Vieitez et al. 2009). WPM medium appears to be suitable for in vitro culture of the Quercus spp. Martinez et al. (2017) stated that WPM medium is good for growth in axillary bud culture of Quercus ilex. Vengadesan & Pijut (2009) also reported that WPM medium was good for shoot cultivation of northern red oak (Quercus rubra). The reason WPM medium is good for culturing Quercus spp. seems to be that the inorganic salt composition is suitable for their culture.

    NAA treatment was most effective for root induction (Fig. 3). In general, IBA is mainly used for in vitro rooting of trees. In our study, specifically, NAA was effective for in vitro rooting. It is not known exactly why NAA is effective for in vitro rooting, but it is presumed to be characteristic for each species. Berardi et al. (1993) found that in vitro rooting in P. calleryana was promoted by auxins, mainly by NAA (2.7 μM).

    They were successfully adjusted to ex vitro condition by acclimatization with transferring to vermiculite and commercial soil. The results of this study show that in vitro cultivation methods can be used for clonal propagation and germplasm conservation of Q. myrsinaefolia superior trees.


    This study was carried out by the Forest Service's Forest Convergence Specialist Training Project (Support for Forest Industry Characterization Research, FTIS Assignment No. 2020186A00-2022-AA02).



    Multiple shoot inductions of Q. myrsinaefolia using cytokinins.


    Effect of culture media on the growth of in vitroQ. myrsinaefolia plantlets.


    Root inductions of Q. myrsinaefolia using auxins in MS medium. (A) Control, (B) NAA, (C) IAA, (D) IBA, and (E) Effect of auxin on root induction rates.


    Effect of soils on the survival rate of in vitro cultured plantlets for acclimatization.

    (A) Vermiculite (B) Commercial soil, (C) Survival rates of in vitro cultured plantlets.


    Effect of cytokinin on induction of Q. myrsinaefolia multiple shoot

    Explants of 3 cm in size were cultured in WPM medium containing cytokinin, and the number of induced stems was counted after 4 and 8 weeks of cultures.
    <sup>*</sup>Primary stems: The stem of first germinated
    <sup>**</sup>Secondary stems: Extra-grown stem after primary stem growth


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