• Journal of Forest and Environmental Science
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• v.23 no.1
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• pp.5-9
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• 2007

Zygotic embryos just after harvest of seeds were immature globular to heart stage. Maturation of zygotic embryos rapidly proceed when zygotic embryos together with small excised parts of endosperm were cultured on 1/3-strength MS solid medium with 2% sucrose, and the zygotic embryos were germinated within two months. Embryogenic callus was formed from the excised segments of germinating zygotic embryos of Eleutherococcus pedunclus on Murashige and Skoog (MS) medium with $4.5{\mu}M$ 2,4-D. The embryogenic callus formation occurred at a low frequency (less than 7%) from hypocotyl segments. The embryogenic calli were maintained on the same medium as primary medium. High frequency somatic embryogenesis was obtained after the cells were transferred to medium lacking 2,4-D. Cotyledonary embryos were germinated and converted into plantlets on medium with $20{\mu}M$ $GA_3$. Embryogenic callus and somatic embryos were produced spontaneously on the surfaces of roots and/or hypocotyls of plantlets. The frequency of embryogenic callus formation was 85% in roots and 34% in hypocotyls. Therefore maintain of cell lines performed very easily. Plantlets with developed epicotyls at more than 3 cm acclimatized at high frequency (89%). While plantlets with small epicotyls (less than 1 cm) were acclimatized at low rate (32%). The soil survived plantlets produced new sprouts after over wintering in the field.

• Journal of Plant Biotechnology
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• v.39 no.1
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• pp.69-74
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• 2012

Using calli of $Muscari$ $armeniacum$ cv. 'Early Giant' that is monocotyledonous ornamental bulb crop with increasing demand in Korea, we carried out current studies to establish an in vitro multiple propagation protocol via somatic embryogenesis. We found that soft pale yellow green calli were induced from leaf explants cultured on all media containing 0.1~3.0 $mg{\cdot}L^{-1}$ auxins such as 1-naphthalene acetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D). However, induced calli showed vigorous growth only when they further transferred on same media containing 2,4-D, 4-amino-3,5,6-tri-chloropicolinic acid (picloram), or 3,6-dichloro-o-anisic acid (dicamba). Although frequency of somatic embryo induction depended on callus source and PGR composition in somatic embryo induction media, somatic embryogenesis was initiated on surface of proliferated calli after transferring on media with no PGR or 0.01 $mg{\cdot}L^{-1}$ NAA co-supplemented with various cytokinins such as $N^6$-benzylaminopurine (BAP). Highest number of embryo at 9.3 per callus clump was obtained when calli which were grown under 0.1 $mg{\cdot}L^{-1}$ picloram supplementation were sub-cultured on medium with 0.01 $mg{\cdot}L^{-1}$ NAA and 0.5 $mg{\cdot}L^{-1}$ BAP. In addition, morphological characteristics of somatic embryo were categorized into following nine phases: globular, biased heart, biased torpedo, early cotyledonary, middle cotyledonary, late cotyledonary, early sprouting, middle sprouting, and late sprouting embryos.

• Korean Journal of Medicinal Crop Science
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• v.15 no.5
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• pp.346-351
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• 2007

An efficient somatic embryogenesis and plant regeneration protocol was developed for Schisandra chinensis Baill, using embryogenic cell suspensions and optimized media conditions. Friable embryogenic callus was induced from cotyledonary leaf and hypocotyl explants of 7 days old seedlings on MS agar medium supplemented with 1.0 to $4.0\;mg\;l^{-1}$ of 2,4-dichlorophenoxyacetic acid (2,4-D). Fast growing and well dispersed embryogenic cell suspensions were developed within two months when embryogenic calli were transferred to MS liquid medium containing $1.0\;mg\;l^{-1}\;2,4-D$. One third strength of MS medium was the best for both overall growth and development of somatic embryos in liquid culture. Over 3400 viable somatic embryos were produced from each 150 ml flask with an initial cell density of 30 mg in 30 ml medium. Germinated somatic embryos developed in liquid medium converted into plantlets after transferred to half-strength MS semi-solid medium. Approximately 90% of the converted plantlets were successfully transplanted to soil and grew into fertile plants.

• 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.2
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• pp.95-99
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• 1995

We have previously established a system for plant regeneration through somatic embryogenesis and Agrobacterium-mediated transformation of Korean ginseng. In this study to produce a fungus-resistant plant, we introduced a bean chitinase gene into ginseng using the transformation system. A binary vector pChi/748 was constructed by introducing the bean basic chitinase gene into EcoRI site of pGA748 which carries the CaMV 35S promoter governing the introduced gene and neomycin phosphotransferase II(NPT-II)gene as a positive selection marker. Cotyledonary explants were cocultured with A. tumefaciens strain LBA4404 harboring the binary vertor pChi/748 for 48 h, and transferred to MS medium supplemented with l mg/L2,4-D,0.1mg/L kinetin, 100 mg/L kanamycin, and 500mg/L carbenicillin. Kanamycin-resistant calli were formed on the cut surface of cotyledonary explants after one month of culture, and subsequently they gave rise to somatic embryos. Upon transfer onto medium containing 1 mg/L each of BA and GA$_3$, most of them converted to plantlets after 5 weeks of culture. The genomic DNA of eight kanamycin-resistant regenerants was subjected to polymerase chain reaction (PCR) using two specific 21-mer oligonucleotides derived from the chitinase gene. PCR-Southern blot analysis confirmed that the chitinase gene was incorporated into six out of the eight regenerants..

• Journal of Plant Biotechnology
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• v.29 no.2
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• pp.135-138
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• 2002

Cotyledonary explants from immature zygotic embryos of each 85 recombinant inbred lines (RILs) were cultured on medium containing MS salts, B5 vitamins, 40 mg$^{-1}$ 2,4-dichlorophenoxyacetic acid and 30 g$^{-1}$ sucrose. Frequency of somatic embryo formation on cotyledonary explants showed in thirty-six lines(＜10%), in thirty-seven lines (11~49%), in nine lines (50~89%), and in three lines(>90%), respectively, The highest frequency (up to 90%) and number (6.36 per cotyledon) of somatic embryos were obtained from lines of KM1010, KM1032 and KM1064. Primary somatic embryos produced from three lines produced numerous secondary somatic embryos on the surfaces, which were subcultured for over one year. Upon transfer to maturation and conversion medium (Komatsuda, 1992), somatic embryos converted to plantlets at a frequency of approximately 25%.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.213-237
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• 1987

In vitro flowering system may minimize the confounded influence of non-floral meristem parts of plants in studying the relationship of a given treatment and flowering responses. We have induced flower buds from plantlets regenerated from zygotic embryo-derived somatic embryos of ginseng, which circumvented the normal 2-year juvenile period before flowering. The result suggests that the adulthood of ginseng root explants in the experiment previously conducted by Chang and Hsing (1980; Nature 284: 341-342) is not prerequired to flowering of plantlets regenerated through somatic embryogenesis. We have also induced flower buds from elongated axillary brandches from cotyledonary nodes by culturing ginseng zygotic embryos, seedlings, and excised cotyledonary nodes. It was found that 6-benzyladenine (BA) supplemented to the medium was essential for flowering, whereas abscisic acid (ABA) was inhibitory. Gibberellic acid(GA3) was also required for flowering when ABA was present with BA in the medium. The results suggest that cytokinins, gibberellins, and inhibitors play primary, permissive, and preventive roles, respective-ly, in the induction of flowering of ginseng. Tran Thanh Van (1980; Int. Rev. Cytol., Suppl. IIA: 175-194) has developed the "thin cell layer system" in which the induction of shoots, roots, or flower buds from epidermal layer explants were controlled by culture conditions and exogenous growth regulators in the medium, Utilizing the thin cell layer system, Meeks-Wagner et al. (1989; The Plant Cell 1: 25-35) have cloned genes specifically expressed during floral evocation. However, the system is too tedious for obtaining a sufficient amount of plant materials for biochmical and molecular biological studies of flowering. We have developed a garlic callus culture system and one obvious advantaging over the thin cell layer system is that an abundant cells committed to develope into flower buds proliferate. When the above cells were compared by two-dimensional gel electrophoresis with those which have just lost the competence for developing into flower buds, a few putative proteins specific to floral evocation were detected. The garlic callus culture system can be further explored for elucidation of the molecular biological mechanism of floral evocation and morphogenesis.hogenesis.

• Journal of The Korean Society of Grassland and Forage Science
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• v.19 no.4
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• pp.297-302
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• 1999

Cotyledonary abnormalities were observed in secondary somatic embryos which were developed from calli cultured on MS medium with various concentrations of 2,4-D. In MS medium containing hormone-free or $0.1mg/{\ell}$ 2,4-D, the frequency of normal embryo formation with two cotyledons were above 57%. According to concentration of 2,4-D increment the frequency of normal embryos were decreased. In MS medium containing $4mg/{\ell}$ 2,4-D, the frequency of normal embryo formation was just 10%. The rate of germination was as follows; 37.7% of somatic embryos had one cotyledon, 85% two cotyledons, 38% three cotyledons, 35% four cotyledons, 25% five cotyledons and 29% trumpet-like cotyledons. About 80% of the embryos with two cotyledons were converted into normal plants, but one, three or four cotyledons were only 6.8~10%. The five or trumpet-like embryos were not developed into normal plants.

• Journal of Korean Society of Forest Science
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• v.97 no.1
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• pp.127-133
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• 2008

This study was conducted to establish the optimal condition for plant regeneration and acclimatization from somatic embryos of Panax ginseng. Cotyledon segments of Panax ginseng produced primary and secondary somatic embryos when cultured on MS and WPM media supplemented with 7% sucrose. To induce plantlet conversion, cotyledonary somatic embryos were cultured on WPM solid medium with $GA_3$ at various concentrations (1~30 mg/L) for 4 weeks. Plantlets were transferred to 1/2 WPM solid medium with $GA_3$ at various concentrations (0~5 mg/L) and 0.5% activated charcoal for shoot and root elongations. Elongated plantlets further developed into well-developed leaf and root system on 1/3 SH medium with 0.5% activated charcoal under ventilation condition for 5 months. The highest survival rate to soil was 75% when plantlets were regenerated on 1/3 SH medium without sucrose under ventilation condition.

• Journal of Plant Biotechnology
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• v.6 no.1
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• pp.45-50
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• 2004

Somatic embryos were induced from immature cotyledons and cultured on a MS medium containing 40mg/L 2,4-D. The maximum induction of embryos was obtained from immature cotyledons in a size of 3-4mm, and the highest frequency was obtained in the induction medium at pH 7.0. For embryo development, embryogenic tissues were transferred to a MSM6AC and MSM6 media. Developing embryos were placed at 27$^{\circ}C$with dim light (20$\mu$$molm^{-2}$$s^{-1}$) provided by cool fluorescent tubes (3-D wavelength light is better than standard light). Somatic embryos were clearly developed from globular stage to cotyledonary stages. The color of embryo may be a useful parameter for estimation of embryo quality. When the embryo becomes mature, embryo will be ready for desiccation in order to induce roots and shoots of embryos.