• 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%.

• Plant Biotechnology Reports
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• v.3 no.3
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• pp.199-203
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• 2009

Rugosa rose (Rosa rugosa) is cultivated as a garden flower and an important genetic resource for the breeding of roses (R. hybrida). This study describes culture conditions for high frequency plant regeneration from zygotic embryo explants via somatic embryogenesis in rugosa rose. Mature zygotic embryo, cotyledon, and radicle explants formed embryogenic calluses at frequencies of 38, 6.7, and 8.8% when cultured on half-strength Murashige and Skoog medium (${\frac{1}{2}}MS$) supplemented with 2.26, 9.05, and $9.05{\mu}M$ 2,4-dichlorophenoxyacetic acid, respectively. Embryogenic calluses produced numerous somatic embryos, which then developed into plantlets on ${\frac{1}{2}}MS$ without growth regulators. Regenerated plantlets were grown to whole plants in a growth chamber.

• 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 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.

• 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.

• Korean Journal of Plant Tissue Culture
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• v.24 no.3
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• pp.129-135
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• 1997

Morphogenetic responses were investigated by culturing embryo and leaf explants of Korean wild type tea plant, Camellia sinensis (L.) O. Kuntze. Induction of direct somatic embryogenesis as well as adventitious and/or axillary shoots was obtained from mature zygotic embryo cultures on Murashige and Skoog (MS) basal medium having 5 to $20\mu\textrm{M}$cytokinin a lone. Morphogenetic response was decreased dramatically by the addition of auxins tested. One hundred percent of induced and isolated shoots formed roots after four weeks of culture on half-strength MS or quarter-strength Schenk and Hildebrandt (SH) media supplemented with $10\mu\textrm{M}$indole-3-butyric acid (IBA). Immature zygotic embryos were shown to be a suitable explant for embryogenic callus formation in the presence of 2, 4-dichlorophenoxyacetic acid(2, 4-D) in basal medium. Mature zygotic embryo originated leaves were used to test their ability for mophogenesis by incorporating plant growth regulators such as IBA, naphthyl-1-acetic acid (NAA), and 6-benzylaminopurine (BAP). Apparently, the morphogenetic responses of the cultured explant sources on the types and/or levels of plant growth regulators tested were observed visually.

• Journal of Plant Biotechnology
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• v.6 no.4
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• pp.213-219
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• 2004

Zygotic embryos were excised from immature and mature seeds of the Himalayan yew, Taxus wallichiana. The embryos germinated precociously when kept in darkness for 5 weeks and developed into full seedlings within 10-12 weeks. The highest rate of embryo germination ($81\%$) was obtained in modified Lloyd & McCown' s woody plant medium containing macro and micronutrients at half strength supplemented with $1\%$ activated charcoal, which supported both the best embryonic growth ($43\%$) and seedling development ($32\%$). However, the supplementation of basal media with kinetin, thidiazuron, 6-benzyl aminopurine or $GA_3$ had no effect on the germination of the embryos. The embryos derived from immature seeds germinated but the frequency of embryonic growth was better in mature seeds. Stratification of seeds effected precocious germination of embryos. Seeds kept at $4^{\circ}C$ for 1 week germinated earlier and at a higher frequency irrespective of the stage of seed maturity, while the germination rate declined with prolonged cold treatment for 1 month at that same temperature. Analysis of taxanes in germinating seedlings revealed that root tissues contained high levels of taxol, 10-deacetyl-baccatin ill and baccatin ill as compared to shoots. Thus embryo culture technique appears to overcome the lengthy dormancy requirement of T. wallichiana seeds.

• Journal of Ginseng Research
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• v.31 no.1
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• pp.14-22
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• 2007

As the zygotic embryo of North American ginseng (Panax quinquefolius L.) matured during stratification over 203 days it grew from 0.75 to 5.2 mm. Embryo excision and culturing on media containing different concentrations of two growth regulators, gibberellic acid ($GA_3$, 1 to 10 ${\mu}M$) and benzyladenine (BA, 1 to 5 ${\mu}M$), during stratification, showed that shoot and root number and the shoot, root and cotyledon length increased with increased stratification time. Gibberellic acid was the more effective growth regulator for increasing shoot and root number and shoot, root and cotyledon lengths. Immature embryos (stratified for up to 63 days) needed growth regulators for further development. Cultures on $GA_3$ at the last culture date (stratified for 203 days) when embryos were mature, produced multiple shoots but there was no effect of $GA_3$ concentration. Benzyladenine inhibited shoot and root growth regardless of embryo stratification. Growth regulators had little effect on cotyledon length of mature embryos. Embryos cultured on $GA_3$ combined with BA were green on all culture dates whereas greening in the control and BA treatments increased with culture date. The BA treatments induced 100% swelling of the embryos on the final culture date while in the control and $GA_3$ treatments there was no swelling. There was little or no curling in the control and BA treatments and a linear decrease in curling with culture date in the $GA_3$ and $GA_3$ + BA treatments.

• Plant Biotechnology Reports
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• v.3 no.4
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• pp.333-340
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• 2009

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with $90.5{\mu}M$ 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with $13.6{\mu}M$ 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.