• Journal of Plant Biotechnology
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• v.30 no.3
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• pp.233-239
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• 2003

We have developed a reliable and high-frequency genetic transformation and regeneration system via somatic embryogensis of Eleutherococcus sessiliflorus. Embryogenic callus obtained from seed were co- cultivated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring genes for intron-$\beta$-glucoronidase(GUS), kanamycin and hygromycin resistance. Following co-cultivation, two types of samples(fine embrogenic calli and early globular embryo clusters) were cultivated on Murashige and Skoog(MS) medium containing 1 mg/L2.4-D for 3day in dark. Transient expression of GUS gene was found to be higher in the early globular embryo clusters than in the embryogenic calli. Also, co-cultivated period affected expression of GUS gene; the best result was obtained when globular embryo clusters were co-cultivated with Agrobacterium for 3 days. Subsequently, this callus transferred to selective MS medium containing 1mg/L2.4-D, 50mg/L kanamycin or/and 30mg/L hygromycin and 300mg/L cefortaxime. These embryogenic calls were subcultured to the same selection medium at every 2 weeks intervals. Approximately 24.5% of the early globular embryos co-cultivated with Agrobacterium for 3days produced kanamycin or/and hygromycin-resistant calli. Transgenic somatic embryos were converted into plantlets in half strength MS medium supplemented with 3mg/L GA$_3$ kanamycin and were confirmed by GUS histochemical assay and polymerase chain reaction analysis. Genomic Southem blot hybridization confirmed the incorporation of NPT II gene into the host genome.

• Journal of Plant Biotechnology
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• v.4 no.1
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• pp.29-32
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• 2002

The ability to form embryiod from callus in satsuma mandarin is low and unstable. In this study, the conditions of embryogenic calli induced from nucellar tissue for promotion of plant regeneration in satsuma mandarin were investigated. The calli of I, II and III line were divided into two sizes of 0.5 mm and 1.0 mm in diameter and two weight gradients of percoll at 40% and 50% though the filter mesh. The frequency of embryo formation from $\phi$ 1.0mm-40% was slightly higher than callus that from others. Adventitious embryoids developed to a globular stage were transferred to regeneration medium. In 'Miyagawa Wase', the embryos from I and II line developed into a heart stage from most of $\phi$ 0.5 mm- 40% and $\phi$1.0 mm-40% calli, but it failed in 'Sugiyama Unshu'. In the cultivar of 'Miyagawa Wase', 63% of adventitious embryos transferred to the regeneration medium developed into the heart stage from the most $\phi$ 1.0 mm-40% calli of I line, but of 'Sugiyama Unshu' failed in some calli condition. The embryoids from two callus lines developed further to shoots and plantlets, while the embriods from III line abnormal failed to regenerate in the cultivar. From these results, it is suggested that the plant regeneration from embryogenic callus in satsuma mandarin could be affected by callus conditions.

• Korean Journal of Plant Tissue Culture
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• v.24 no.6
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• pp.369-374
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• 1997

Immature flower buds of 'Desio' carnation were cultured on MS agar medium supplemented with 1 ㎎/L 2,L-D. Embryogenic calli were formed from 5-10% of the buds less than 20 ㎜ in length, but only non-embryogenic calli were produced from explants of shoot apex leaf, internode, and flowere buds larger than 20 ㎜. The same method was applied to 16 cultivars of cut Sower carnation and embryogenic calli were obtained in 7 cultivars. Several embryogenic callus lines were selected and maintained through subcultures over 120 weeks without loss of embryogenic competence. The embryogenic cultures were also proliferated rapidly in liquid agitation cultures using MS medium supplemented with 1mg/L 2,4-D. Numerous embryos were formed on the periphery of the cell aggregates upon transfer to auxin-free MS agar medium. Plantlets were transplanted in potting soil and grown to bloom in six months.

• Journal of Plant Biotechnology
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• v.4 no.4
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• pp.163-170
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• 2002

Radiation induced and somaclonal variations were investigated in the regenerates from gamma irradiated and controlled embryogenic callus (EC) of sweetpotato cvs., Yulmi and White Star by morphological, RAPD and AFLP analysis. Most (approx. 90%) of the EC produced somatic embryos developed into plantlets after being transferred to the auxin-free medium. The frequency of morphological variants derived from the irradiated callus ranged from 3 to 7.8% compared to 0.1-1.1% of that derived from the non-irradiated. Morphological variants were selected from the regenerates and analyzed by RAPD and AFLP procedures. RAPD polymorphisms of Yulmi and White Star regenerates from irradiated calli were 8.8% and 6.1%, respectively. However, the polymerphisms among regenerates from the non-irradiation treatment in these two cultivars were non-detectable and 3%, respectively. AFLP polymorphisms of Yulmi and White Star regenerates from irradiated calli were 29.9% and 28.6%, respectively. while the frequencies for those form non-irradiated calli were 8.5% and 5.6%, respectively. Both the control plants and variants from the nonirradiated were clustered together, while variants from irradiated were separated from the group by Nearest-Neighbor-Interchange Branch Swapping Abbreviation: EC (Embryogenic callus), AFLP (Amplified Fragment Length Polymorphism), RAPD (Random amplified polymorphic DNA)

• Korean Journal of Plant Tissue Culture
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• v.26 no.2
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• pp.121-126
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• 1999

Calli were induced from the petiole explants of Pimpinella brachycarpa on MS medium supplemented with 0.5 mg/L 2,4-D and 0.1 mg/L BA after four weeks of culture. Compact clusters of small and dense cells among these calli were selected and suspension-cultured as the source of embryogenic calli. When transferred to MS medium with 0.1 mg/L NAA, the suspension-cultured cells grew to embryogenic callus. Somatic embryos derived from these embryogenic calli developed into plantlets. The cDNA library was constructed in the embryogenic callus and in order to screen the cDNA library, these cDNAs were plated at a density 1.5 $\times$ 10^5 plaques per 15 cm petridish. Among 19 clones showing preferential hybridization with petiole HD-Zip gene, five clones were obtained after second screening. Four clones among them, were highly homologous to P. brachycarpa shoot-tip Phz4 gene, but one clone, Phc5 was about 1.5 kb which has an extra 163 bp to 5' upstream of Phz4. The Phc5 was 1,531 bp containing poly A tails of 18 bases. ATG start codon for Phc5, was located at position 284 with an open reading frame of 906 by which encodes a polypeptide of 302 amino acids. The Phc5 protein revealed that the polypeptides between 135 and 195 contain a homeodomain as the `leucine zipper' motif.

• Journal of Plant Biotechnology
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• v.30 no.4
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• pp.341-346
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• 2003

In an effort to optimize tissue culture conditions for genetic transformation of orchardgrass (Dactylis glomerata L.), an efficient and high-frequency plant regeneration system from seed-derived calli was established. Embryogenic calli induced on MS medium containing 3mg/L 2,4-D and 0.1mg/L BA had significantly improved regeneration ability. Plant regeneration rate was 92％ when embryogenic calli were cultured on N6 medium supplemented with 1mg/L 2,4-D and 3mg/L BA. Among three kinds of medium, MS and N6 medium were optimal for embryogenic callus induction and plant regeneration, respectively. Ho difference in callus induction frequency was observed among four cultivars of orchardgrass, however, "Roughrider" cultivar showed higher regenerability with the frequency of 61％. Addition of maltose to the regeneration medium as a carbon source dramatically increased regeneration frequency up to 69％. A short tissue culture period and high-frequency regeneration system would be beneficial for molecular breeding of orchardgrass through genetic transformation.

• Journal of Plant Biotechnology
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• v.2 no.2
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• pp.109-113
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• 2000

Somatic hybrids were produced by electrofusion between embryogenic callus protoplasts of 'Syougun' mandarin and leaf protoplasts of grapefruit. Hybridity of the two plants was confirmed by leaf morphological characteristics and random amplified polymorphic DNA (RAPD) analysis. The cpDNA analysis using PCR-RFLP could not distinguish those of both parents. These plants showed normal growth and had chromosome number of 27. These unexpected triploid somatic hybrids might be derived from fused cells between diaploid protoplast of embryogenic calli and diploid protoplast of leaf, because polysomaty, a mixture of haploid cells and diploid cells was observed in the lactose medium-pretreated embryogenic calli of 'Syougun' by flow cytomehy analysis.

• Journal of The Korean Society of Grassland and Forage Science
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• v.20 no.1
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• pp.13-18
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• 2000

An efficient method for Agrobacterium-mediated transformation of forage crop alfalfa (Medicago sativa L.) was established using secondary somatic embryogenic calli. Agrobacterium tumefaciens strain EHAlOl and a binary vector pIG121-Hm which has selection markers for kanamycin and hygromycin have been shown to be an efticient materials for alfalfa transformation. The secondary somatic embryogenic calli originated from hypocotyl explants of alfalfa were efficient infection materials for Agrobacterium EHAlOl and normally germinated into plantlets. The introduced gene (GUS) was constitutively expressed in all tissues of transgenic alfalfa with different expression levels. These results indicate that the use of pIG121-Hm vector, Agrobacterium EHAlOl and improved culture system of callus facilitate the transformation of alfalfa. (Key words : Agrobacterium, Alfalfa, Gene transfer, Transformation)

• Journal of Plant Biotechnology
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• v.31 no.3
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• pp.219-223
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• 2004

Embryogenic calli were induced from petioles of Aralia elata on MS solid medium supplemented with 1.0 mg/L 2,4-D. When embryogenic calli were transferred to MS liquid medium supplemented with 1.0 mg/L 2,4-D, embryogenic cells and embryogenic cell clusters were developed after 2 weeks of culture. Embryogenic cells were filtered through a 250 ${\mu}{\textrm}{m}$ sieve and the passed cells were proliferated and maintained in MS liquid medium supplemented with 1.0 mg/L 2,4-D. Embryogenic cell clusters entrapped on the sieve were transferred to 1/2 MS liquid medium without plant growth regulators, globular-shaped embryos were developed from embryogenic cell clusters after 2 weeks of culture. Numerous early stage somatic embryos could be developed to heart-shaped, torpedo-shaped, cotyledonary embryos and plantlets in 5 L bioreactor. Above results suggest that effective somatic embryo proliferation can be achieved via bioreactor culture systems in Aralia elata.

• Journal of Plant Biology
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• v.36 no.4
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• pp.377-382
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• 1993

Rice (Oryza saliva L.) calli containing both embryogenic callus (EC) and non embryogenic callus (NEC) regions were initiated from the mature seed on MS medium supplemented with 2.0 mg/L 2,4-D, 0.5 mg/L kinetin. The calli were developed into two callus type which can be distinguished by visual examination depending on color and appearance. In order to illucidate the polypeptide composition between EC and NEC, the total protein extracted from two types of callus was analysed by electrophoresis. By one-dimesional anlaysis of SDS-PAGE and Isoelectric focusing, several protein bands showed quantitative and qualitative difference in each type of callus. The further analysis of the total protein with two-dimensional electrophoresis showed at least 20 EC specific protein and 10 NE specific protein. Also 3 specific protein spots showing micro heterogeneity of 90, 65, 50 kD were detected in EC, while a series of acidic heterologous protein spots were visualized in NEC.in NEC.