J Plant Biotechnol (2018) 45:347–356 ISSN 1229-2818 (Print) DOI:https://doi.org/10.5010/JPB.2018.45.4.347 ISSN 2384-1397 (Online) Research Article Optimization of factors influencing in vitro immature seed germination in Chionanthus retusus Khin Yae Kyi Tar ･ Aung Htay Naing ･ Trinh Ngoc Ai ･ Mi Young Chung ･ Chang Kil Kim Received: 14 September 2018 / Revised: 19 November 2018 / Accepted: 6 December 2018 ⓒ Korean Society for Plant Biotechnology Abstract Chionanthus retusus is a small deciduous tree highest germination (95.37%). These results of this study that is widely used in landscaping due to its beautiful white could be an open ground for development of an efficient spring flowers and ornamental value. Conventional propagation protocol for commercial production of the ornamental tree. through seeds requires one to two years of breaking dormancy. The objective of this study was to determine the Keywords Chionanthus retusus, embryo culture, in vitro conditions of in vitro germination in C. retusus. In vitro germination embryo culture was carried out to investigate the effects of six factors: basal media (McCown Woody Plant Medium (WPM) and Murashige and Skoog (MS)); plant growth Introduction regulators (different combinations and concentrations of naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BA), The Chinese fringe tree (Chionanthus retusus) is a small and gibberellic acid (GA₃)); embryo age (collected weekly deciduous tree in the family Oleaceae and is native to China, beginning 36 days after fruit setting); low temperature Korea, Japan, and Taiwan (Nicholson 1990). In Korea, the pretreatment (storing 4°C for 1, 2, 3, and 4 weeks); coconut Forest Research Institute reports that C. retusus can be found additives (100, 200, and 300 ml·L-1); and genotype (grouping growing near sea level to over 900 meters inland (Nicholson plants depending on their flowering nature). The basal 1990). The genus Chionanthus consists of nearly 100 species -1-1 distributed worldwide (Lombardi 2006), the majority of which medium used in this study was WPM with 2 mg·L GA₃, -1 -1 are tropical to subtropical and native to parts of Africa, Asia, 20 g·L sucrose, and 6 g·L Agar. WPM medium mixed the Americas, and Australia (Green 1994). The morphological with GA₃, resulted in higher germination rate as compared to characters and reproductive processes of the Chinese fringe when using a combination of auxin and cytokinin. GA₃ at 2 tree are similar to those of both C. virginicus and C. mg·L-1 was the most effective of all combinations and pygmaeus, which are prized for their attractive ornamental concentrations of PGRs. WPM medium with 2 mg·L-1 GA₃ features (Fagan, A. E. and Dirr, M. A. 1980). The growth of C. resulted in better and faster germination (75.93%). Embryos retusus is very slow, usually growing 4 to 10 inches per year collected at 57 days after fruit setting had the highest percent and reaching 12 inches in rich, moist, well-fertilized soil of germinated seeds (87.04%) while low-temperature (Gilman and Watson 1993). It is used widely in landscaping pretreatment of fruits at 4°C for two weeks produced the owing to its ornamental value. Its leaves have been used in folk medicine for the treatment of diarrhea, palsy, and K. Y. K. Tar ･ A. H. Naing ･ C. K. Kim () stomachache (Kwak, et al. 2009). In China, the flowers are Department of Horticultural Science, Kyungpook National used to prepare tea owing to their content of various University, Daegu, Republic of Korea e-mail: [email protected] flavonoids and triterpenoids and for their medicinal value; the young shoots and leaves are used in cooking and young leaves T. N. Ai School of Agriculture and Aquaculture, Tra Vinh University, Trà are used as an alternative for tea (Nicholson 1990). C. retusus Vinh, Vietnam can grow well in full sun to partial shade, with moderate summer drought, and in a wide range of soil conditions (Dirr, M. Y. Chung Department of Agricultural Education, Suncheon National 1998) (Gilman and Watson 1993). It can reach 25 m (80 feet) University, Suncheon, Korea This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 348 J Plant Biotechnol (2018) 45:347–356 in height and 70 cm (2 feet) in girth under full sun and high radical protrusion and dormancy breaking. In other words, moisture conditions (Kurata 1973). The flowering time is protrusion of the radical of the embryo occurs slowly under from late April through mid-May, and full, showy, white favorable temperatures prior to cold treatment, which can flowers are produced. Fruits are produced at the end of May. cause the embryo to be completely non-dormant. Chionanthus species have double dormancy and are conven- Embryo culture has been used to overcome seed dormancy tionally propagated via warm/cold stratification of the seed. in many other species (Arrillaga et al. 1992; Bridgen 1994). They are considered difficult to propagate vegetatively Immature embryo culture is the culturing of zygotic embryos (Nicholson 1990) because of their lack of rooting. Other than cut off from ovules and seeds under sterile conditions in for Chionanthus, the vegetative propagation of other mem- growth regulators. Many factors influence on the successful bers of Oleaceae is nearly impossible, for example with stem development of an embryo, important factors include genotype, cuttings of Fraxinus spp. (ash) (Dirr and Heuser 1987). explant, embryo age, composition of basic growth media, Rooting has been attempted in softwood cuttings collected growth regulators, light intensity and quality, photoperiod, in June to mid-July using 1.0% indole-3-butyric acid (IBA), temperature and endogenous factors (Umehara et al. 2007; but success was only around 50% (Dirr and Heuser 1987). Polanco and Ruiz 2001). Complex media supplemented with Around 95% rooting has been achieved from stem cuttings a combination of vitamins, amino acids, growth hormones collected in mid-July using 8000 ppm IBA, but these results and, in some cases, natural extracts, such as tomato juice and are difficult to consistently replicate (Dirr 1998). Attempts to coconut milk, are required for the development of young initiate rooting from hardwood cuttings and root cuttings embryos (Bridgen 1994). treated with IBA (1000 ppm and 8000 ppm) were unsuccessful A period of cold, moist conditions (prechill) is required for for C. retusus, C. virginicus, and C. pygmaeus by Eads, Amanda the germination of certain seeds (Feghahati and Reese 1994). L. Successful root formation depends on the combination of Seeds with morphophysiological dormancy require either cutting age and/or the extent of lignification in the cuttings treatment with GA3, abscisic acid (ABA), cytokinins and (Cameron, et al. 2003). An experiment comparing the rooting auxins (Seo et al. 2011), or a combination of warm stratification behavior of C. retusus and C. virginicus was conducted under and moist-chilling to break dormancy (Hilhorst 2011) and mist, using half peat and half perlite medium by Arnold initiate the fast multiplication and growth of cells, followed Arboretum Propagator Jack Alexander (Nicholson 1990). No by the protrusion of radicles. However, studies on embryo root formations were found, even when hormone treatments culture in this species are rare. Charlotte R. Chan (1999) reported were used in C. virginicus. Rooting occurred at 30% in C. that embryo culture in C. virginicus started germination within retusus using 1% indolebutyric acid in a solution of 50% 7 to 10 days and traditional seed propagation (warm /cold ethanol and 59% water. Therefore, seed propagation is the stratification) required 8 months for radicle protrusion. The most reliable propagation technique for Chionanthus, even size of 10-month-old cultured plants was comparable with the though the seeds have double dormancy (Nicholson 1990). size of 2-year-old traditionally grown plants. In 2009, I-Ching Carpenter, W. J. observed that endocarp-removed seeds of C. Huang reported that seed dormancy was broken in C. retusus virginicus L. needed 10 weeks (26%), while mechanically and by culturing the embryo on Woody Plant Medium (WPM) acid scarified seeds needed 18 weeks (2%) and 14 weeks containing 2 mg/L GA3 (Huang et al. 2009). (5%), respectively, to germinate to 50% final germination. Therefore, in this study, we investigated several conditions The highest 50% of germination (82%) was observed after 4.6 for the in vitro germination of C. retusus embryos, including weeks at 25°C in endocarp-removed seeds pretreated with medium composition, low-temperature pre-storage, embryo 1000 ppm GA3 for 6 hours. C. retusus showed an unusual age, plant growth regulators, and genotype. The objective of form of epicotyl dormancy, similar to that in Davidia this study was to identify the conditions most suitable for in involucrata (dove-tree), Paeonia suffruticosa, and Aesculus vitro germination in C. retusus. parviflora (bottlebrush buckeye). In epicotyle dormancy, the root comes out during the first year after sowing but shoot appearance is extended to the next year after the seed has been Materials and Methods through a cold winter (Chien et al. 2004). According to Baskin’s report (Baskin and Baskin 2004), Chionanthus seeds, Immature C. retusus fruits were collected in July from the which show epicotyl dormancy, belong to the class of seeds campus of Kyungpook National University, Daegu, and that show deep morphophysiological dormancy, i.e., seeds Republic of Korea. Fruits were washed with distilled water must be exposed to warm and then cold stratification for three times and put in 70% ethanol for 1 minute. Next, fruits J Plant Biotechnol (2018) 45:347–356 349 were rinsed in 1% sodium hypochlorite solution (10 mL·L-1) EM4, 57 days after fruit set.