• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.223-227
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• 2015

Embryogenic callus (EC) was created from mature embryos of Larix kaempferi. With the mature embryos, keeping the culture in dark conditions throughout the experiment (38.2%) seemed to give better results than exposing them to 16 h light ($25{\mu}Em^{-2}s^{-1}$) for the first week (21.9%). EC was obtained most frequently from Quoirin and Lepoivre (LP) mediums with 1.0 mg/L 4-amino-3,5,6-trichloropicolinic acid (Picloram), plus 1.0 mg/L benzyladenine (BA) (62.8%) or Litvay's medium (LM) containing 1.0 mg/L p-chlorophenoxyacetic acid (pCPA) plus 1.0 mg/L BA (62.8%) treatment. In both cases, best results were obtained when zygotic embryos were cultured in darkness. As for the effective sucrose concentration on initiation of EC, 29.2 mM sucrose (38.6%) gave the best results.

• Journal of Plant Biotechnology
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• v.7 no.1
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• pp.75-80
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• 2005

Eleutherococcus senticosus (also called Acanthopanax senticosus), belonging to Araliaceae family, has been used as an important medicinal woody plant. Mature seeds of Eleutherococcus senticosus have rudimentary (extremely immature) zygotic embryos and require a long-term stratification for about 18 months to induce germination. Here, through the methods of endosperm removal and other exogenous treatments, we investigated the factors for inducing rudimentary embryos by in vitro culture, Rudimentary zygotic embryos in seeds were at globular to heart-shaped stage at about $250{\mu}m$ in length just after harvest of fruits. When the seeds without testa were cultured on 1/2 MS (Murashige and Skoog 1962) medium, they did not germinate regardless of medium and sucrose concentrations but the removal of endosperm tissue markedly stimulated the growth of rudimentary zygotic embryos. The embryo reached ear-lier maturation, once when the endosperm surrounding the rudimentary embryos was removed. Rudimentary zygotic embryos developed cotyledons within 3 weeks of culture after endosperm emoval. However, post-mature zygotic embryos failed to germinate though they were morphologically normal, indicating another dormancy of embryos. $GA_3\;(2.0\;\cal{mg/L})$ and/or charcoal ($0.2\%$) treatment rapidly enhanced the germination of zygotic embryos. These results suggest that E. senticosus seeds have double dormancy; i. e. morphological rudimentary dormancy influenced by surrounding endosperm and physiological dormancy after post-maturation of zygotic embryos. Based on the above findings, we established the rapid germination of rudimentary zygotic embryos by in vitro culture of excised seeds with endosperm removal and $GA_3$ treatment.

• Journal of Plant Biology
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• v.37 no.3
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• pp.365-370
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• 1994

Excised cotyledon segments of ginseng zygotic embryos at various developmental stages were cultured on MS basal medium from which somatic embryos were directly induced. The frequency of somatic embryo formation on the segments declined with the advancing zygotic embryo maturity. All of the cells in the cotyledons of immature zygotic embryos were smaller and more densely cytoplasmic than those in mature embryos. Histological examinations revealed that the poly-somatic embryos formed on immature embryos were of multi-cell originand derived from the epidermal and subepidermal cell layers. However, in the cotyledon of germinating zygotic embryos, only theepidermal cells were densely cytoplasmic and singularly competent to develop into somatic embryos resulting into single embryos at a frequency of 100%.

• Journal of Plant Biotechnology
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• v.45 no.4
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• pp.333-339
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• 2018

The mature zygotic embryos of the Hevea brasiliensis were cryopreserved through the use of the vitrification and encapsulation/dehydration techniques. In all the experiments, the zygotic embryos were pre-cultured for three days in the MS medium supplemented with 0.3 M sucrose before they were used for the cryopreservation technique. In the vitrification procedure, the effect of the plant vitrification solutions (PVS2 and PVS3) and exposure time were studied. The highest survival rate (88.87%) and regrowth (66.33%) were achieved when the precultured zygotic embryos were incubated in a loading solution for 20 minutes at $0^{\circ}C$. They were subsequently exposed to PVS2 for 120 minutes at $0^{\circ}C$ and plunged directly into liquid nitrogen. Cryopreservation by the encapsulation-dehydration method was successfully done by leaving the encapsulated zygotic embryos in a laminar flow for 4 hours prior to plunging into a LN. The survival rate and regrowth of the encapsulated zygotic embryos were 37.50% and 27.98%, respectively. The cryopreserved zygotic embryos were able to develop into whole plants.

• Journal of Plant Biotechnology
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• v.35 no.3
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• pp.195-201
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• 2008

A simplified technique that cryoprotects zygotic embryos by desiccation was developed for germplasm conservation of wild yam species(Dioscorea spp.) in Korea. Comparative studies with three other cryogenic techniques were conducted. The maximum survival of zygotic embryos were achieved at a frequency of 96.6% when embryos were cryopreserved by the desiccation method. For the successful cryopreservation of yam zygotic embryos, those that were excised from immature/mature seeds were dried in the air stream of a laminar flow cabinet for 30 min at room temperature and then directly immersed in liquid nitrogen.

• Journal of Plant Biology
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• v.39 no.2
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• pp.127-135
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• 1996

An efficient system of somatic embryogenesis was established for the red pepper plant (Capsicum annuum L. cv. Nokkwang) usign immature zygotic embryos. The size of the immature zygotic embryos and the concentrations of 2, 4-D and sucrose were found to be critical. Somatic embryos were induced via callus or directly from explants and regenerated into plantlets successfully. When zygotic embryos 1~2 mm long were cultured on the modified Murashige-Skoong (MS) medium supplemented with 2 mg/L 2, 4-D for 3 weeks in the dark, somatic embryos were induced directly from the apical region of zygotic embryos with the highest frequency being approximately 90%. To mature the somatic embryos, ABA and an ethylene inhibitor AgNO3 were used. The highest frequency of shoot regeneration (25% in each) resulted at 2$\mu$M ABA or 20$\mu$M AgNO3 treatment at rates 3.7 and 1.6 times control, respectively. Shoots developed mainly from the cotyledonary node on CoCl2-containing medium, and from the upper side of cotyledon on medium containing AgNO3 while the embryos on the control medium produced shoots from both the cotyledonary node and the upper region of cotyledons both at frequencies of 50%. Indirect somatic embryogenesis via callus was induced at an efficiency of approximately 10% with zygotic embryos 3~4 mm long cultured on MS medium containing 5~10 mg/L, 2, 4-D for 5~7 weeks under a continuous light condition. The plants regenerated from the somatic embryos were morphologically normal. Using scanning electron microscopy, the direct and indirect somatic embryogeneses were observed to follow the globular, heart and torpedo stages, similar to zygotic embryogenesis. Also, suspensors appeared in the early globular and ovoid-shaped late globular embryos during indirect somatic embryogenesis.

• Journal of Plant Biology
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• v.32 no.3
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• pp.145-150
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• 1989

Mature zygotic embryos dissected from ginseng(Panax ginseng C. A. Meyer) seeds were cultured on Murashige and Skoog's (MS) medium containing various concentrations of 2, 4-dichlorophenoxyacetic acid(2, 4-D) and kinetin. Somatic embryos were induced directly from cotyledonary tissue or from intervening callus. The induction frequency of somatic embryos was up to 55%. Upon transfer to half-strength MS medium supplemented with 1 mg/1 6-benzyladenine(BA) and 1 mg/1 GA3, most somatic embryos developed into plantlets. Over 50% of the plantlets flowered after 4 weeks of culture and then a few bore immature fruits in vitro. Therefore, it is suggested that the juvenility of the ginseng tissue which give rise to somatic embryos does not interfere with in vitro flowering of their regenerated plantlets.

• Korean Journal of Plant Tissue Culture
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• v.22 no.3
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• pp.157-164
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• 1995

Intact mature zygotic embryos or their excised cotyledons of ginseng, were cultured on media containing various growth regulators such as auxin (2,4D, IAA) and cytokinin(BAP kinetin). In the culture of intact zygotic embryos, auxin inhibited germination but cytokinin did not Somatic embryogenesis occurred only from those of ungerminated embryos. In the culture of cotyledon segment, medium without growth regulators was the most appropriate to somatic embryogenesis. Somatic embryos were produced sporadically over the surfaces of zygotic embryos on medium containing auxin, while on medium without growth regulators, or media containing cytokinin, somatic embryos formed only on the proximal region of cotyledon. on medium containing 2,4-D, somatic embryos originated from multiple cells which comprised epidermal and subepidermal layers of cotyledon, which resulted in poly-somatic embryogenesis. When these somatic embryos were cultured on the same medium, the primary somatic embryos procured secondary embryos, which arose from epidermal or subepidermal single cells.

• Proceedings of the Botanical Society of Korea Conference
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• 1999.07a
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• pp.85-89
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• 1999

Somatic embryogendesis is one of good examples of the basic research for plant embryo development as well as an important technique for plant biotechnology. This paper describes the direct somatic embryogenesis from zygotic embryos of Panax ginseng is reversely related to normal axis growth of zygotic embryos by the experiment of various chemical treatments. Under the normal growth condition, the apical tips of embryo axis produced an agar-diffusible substance, which suppressed somatic embryo development from cotyledons. Although the cells of zygotic embryos were released from the restraint of embryo axis, various factors were still involved for somatic embryo development. Electron microscopic observation revealed that the ultrastructure of cells of cotyledon epidermis markedly changed before initiation of embryonic cell division, probably indicating reprogramming events into the cells embryogenically determined state. Polar accumulation of endogenous auxin or cell-cell isolation by plasmolysis pre-treatment is the strong inducer for the somatic embryo development. The cells for the process of somatic embryogenesis might be determined by the physiological conditions fo explants and medium compositions. Direct somatic embryos from cotyledons fo ginseng were originated eithrer from single or multiple cells. The different cellular origin of somatic embryos was originated either from single or multiple cell. The different cellular origin of somatic embryos was depended on various developmental stages of cotyledons. Immature meristematic cotyledons produced multiple cell-derived somatic embryos, which developed into multiple embryos. While fully mature cotyledons produced single cell-derived single embryos with independent state. Plasmolysis pretreatment of cotyledons strongly enhanced single cell-derived somatic embryogenesis. Single embryos were converted into normal plantlets with shoot and roots, while multiple embryos were converted into only multiple shoots. GA3 or a chilling treatment was prerequisite for germination and plant conversion. Low concentration of ammonium ion in medium was necessary for balanced growth of root and shoot of plantlets. Therefore, using above procedures, successful plant regeneration of ginseng was accomplished through direct single embryogenesis, which makes it possible to produce genetically transformed ginseng efficently.

• Journal of Ginseng Research
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• v.23 no.2 s.54
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• pp.74-80
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• 1999

Excised cotyledon segments of ginseng zygotic embryos cultured on MS basal medium without growth regulators produced somatic embryos near the basal excised portion at a high frequency. The frequency of somatic embryo formation on the segments declined along with advancing zygotic embryo maturity. In immature cotyledons, all the cells of the epidermis and subepidermis were smaller and more densely cytoplasmic than those in mature cotyledons, and from which multiple cells participated in embryogenic division to form somatic embryos with multiple cotyledons and fasciated radicles (poly-embryos). But in germinating cotyledons, only the epidermal cells were densely cytoplasmic and singularly competent to develop into somatic embryos resulting in single-embryos with closed radicles. This result means that the origin and development of somatic embryos is determined according to whether the cells participating in embryonic division are in a single state or a massive state relative to cotyledon maturity.