Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576 S505

[P-P&F.88]

Molecular identification of medically important Zanthoxylumschinifolium using ribosomal DNA internal transcribed spacers

Yan-Lin Sun 1, Wan-Geun Park 2, Oh-Woung Kwon 3, Soon-KwanHong 1,4,∗

1 Department of Bio-Health Technology, Kangwon National University,Chuncheon, Republic of Korea2 Department of Forest Resources, Kangwon National University,Chuncheon, Republic of Korea3 Korea Forest Seed & Variety Center, Suanbo, Chungju, Republic ofKorea4 Division of Biomedical Technology, College of Biomedical Science,Chuncheon, Republic of KoreaKeywords: Zanthoxylum schinifolium; Molecular identification;Phylogenetic relationship; ITS region

Molecular approaches are now being developed to provide amore rapid and objective identification compared to traditionalphenotypic methods. Nuclear ribosomal DNA (nrDNA) targets,especially internal transcribed spaces 1 and 2 (ITS1 and ITS2), havebeen widely used for molecular identification of some plants andfungi.

We therefore conducted an investigation in the identificationof fifth medically important Zanthoxylum schinifolium ecotypesusing the common primers of the ITS region to compare with thesequence variance of the ITS1, ITS2 and total ITS region.

About 620 bp fragments were obtained in this study and thesequences of the PCR products were tested. The sequence length,G+C content (%), DNA alignment and pariwise nucleotide compar-isons demonstrated 98.8 to 100% sequence identities in the total ITSregion, 98.3 to 100% identities in the ITS1 region, and 99.5 to 100%in the ITS2 region. Comparative analysis using GenBank referencedata showed that the exclusive reported data showed 100% iden-tities with BEMR, CWDO, HCDC, JDGG and GJGD in the ITS1 regionand 100% identities with thirteen ecotypes except BEMR and GRDGin the ITS2 region. The fifth different ecotypes were classified intofive groups, and the identification of medically important Z. schini-folium was highly improved due to the augmentation of our currentITS sequences.

The emergence of sequence identification with a BLAST simi-larity search connected to public databases has resolved severalexperimental and taxonomic constraints. Thus, other molecularidentification such as ndhF and EST should be done to explore thepossibility of the multiple variety of sequence. This work not onlyprovides more resources of ITS sequence in Z. schinifolium, but dis-tinguishes five groups from fifth different ecotypes, which makesit possible to elucidate the phylogenetic relationships of Z. schini-folium species.

doi:10.1016/j.jbiotec.2010.09.791

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Molecular identification of Zanthoxylum piperitum by theinternal transcribed spacers as targets using newly designedspecies-specific primers

Yan-Lin Sun 1, Wan-Geun Park 2, Oh-Woung Kwon 3, Soon-KwanHong 1,4,∗

1 Department of Plant Biotechnology, Kangwon National University,Chuncheon, Republic of Korea2 Department of Forest Sources, Kangwon National University, Chun-cheon, Republic of Korea3 Korea Forest Seed & Variety Center, Suanbo, Chungju, Republic ofKorea4 Institute of Bioscience and Biotechnology, Kangwon National Univer-sity, Chuncheon, Republic of KoreaKeywords: Molecular identification; ITS region; Zanthoxylum piper-itum

The genus Zanthoxylum belongs to the family Rutaceae, hav-ing significant medical importance. The present investigation wasundertaken with an aim to isolate the total internal transcribedspacers (ITS) region of the nuclear ribosomal DNA (nrDNA) in Zan-thoxylum piperitum and distinguish from Zanthoxylum sichinifoliumusing nrDNA ITS sequence as markers.

The universal ITS5/ITS4 primer pair could produce PCR amplifi-cation for some plant species including Z. schinifolium and fungi, butonly a small amount of PCR product for Z. piperitum. Species-specificprimers able to amplify the total ITS region sequence, named ITS1-YL1 and ITS1-YL2, have therefore been developed and used for theidentification of Z. piperitum.

We have investigated the specificity of these primers against 21ecotypes of Z. piperitum and 6 of Z. schinifolium. Our results showedthat ITS1-YL1 or ITS1-YL2, when paired with the universal primerITS4, efficiently amplified DNA from Z. piperitum, but not Z. schini-folium. ITS1-YL1/ITS4 or ITS1-YL2/ITS4 preferential amplificationwas shown to be particularly useful for detection and distributionof Z. piperitum from other plant species, especially Z. schinifolium.These primers can be used to study the structure of Rutaceae familyby enhanced specificity, and the identification using ITS region willhelp facilitate alignment of sequences for phylogenetic analysis inintraspecies population of genus Zanthoxylum.

In conclusion, this work not only provides more resources ofITS sequence in Z. piperitum using newly designed species-specificprimers, but also distinguishes from Z. schinifolium, which makes itpossible to elucidate the phylogenetic relationships of genus Zan-thoxylum.

doi:10.1016/j.jbiotec.2010.09.792

[P-P&F.90]

Biomass manipulation of poplars using a gene for cytokininbiosynthesis

Y.I. Choi 1,2,∗, E.W. Noh 1,2, M.S. Han 1,2, J.S. Lee 1,2, M.S. Kim 1,2,K.S. Choi 1,2

1 Korea Forest Research Institute, Republic of Korea2 ChungNam National University, Republic of KoreaKeywords: Poplar; Biomass; Transformaion; Cytokinin

Poplars are fast growing and high biomass yielding trees forintensive cultivation with short rotation. To further improve theproductivity, we attempted to modulate cellular hormone contentto the level that is low enough to resume normal field growth

S506 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

but still high enough to stimulate biomass production. For this,a bacterial trans-zeatin secretion gene (tzs) was modified to becontrolled by either AUX promoter of A. rhizogenes or CaMV 35S.Twenty transgenic poplar (Populus alba x P. tremula var. glandu-losa) lines have been generated by Agrobacterium-stem segmentcocultivation. They were screened for their expression by north-ern and then by cellular trans-zeatin level. Northern blot revealedthat total tzs mRNA by AUX promoter was approximately 200 to 300folds lower than that by 35S promoter. Nevertheless, cellular trans-zeatin level ranged from 0.01 to 0.02 (ng/g/F.W) for nontransgeniccontrol, 0.05 to 0.22 (ng/g/F.W)for pAUX-tzs lines and 0.32 to 0.60(ng/g/F.W) for p35S-tzs lines, respectively. Unlike p35S-tzs lineswhich could not be transplanted outside due to lack of rooting, allthe pAUX-tzs poplars rooted well. However, the cellular cytokininlevel varied even among pAUX-tzs lines. The relationship betweenhormone content and growth performance was compared with cut-tings in the nursery for 2 years. In the first year, two transgenicclones containing low level (0.02 ng/g/F.W) trans-zeatin not onlyperformed better in height growth but also produced more axillaryshoots and thus resulted 1.3 times more biomass than did nontrans-genic control. In the second year, the same lines produced 2.1 timesmore biomass than nontramsgenic control. However, two pAUX-tzsclones containing high level (0.2 or higher)(ng/g/F.W) trans-zeatinproduced too many thin axillary shoots and showed suppressedheight growth leading to very low biomass production. The resultssuggest that for maximal biomass growth, the cellular trans-zeatincontent should be no higher than 0.03(ng/g/F.W).

doi:10.1016/j.jbiotec.2010.09.793

[P-P&F.91]

Physicochemical study of two active substancies extracted fromleaves of Medicago sativa and evaluation of its antibacterialactivity

M. Bouzeraa-Bessila ∗, A. Djahoudi, C. Bouzaata

Annaba University Biology, AlgeriaKeywords: Active substances; Physico-chemical; Analy-sis, Flavonoids; Saponins, Medicago sativa; Leaves extracts;Antibacterial activity

In spite of the manufacture of synthetic drugs emerged in abun-dance and great reputation, medicinal plants inexhaustible sourceof active compounds, are nowadays the main source and basis ofnumerous pharmaceutical specialties. However, large number ofother plant species occurring in nature, in enormous quantities andwith interesting therapeutic properties, remains untapped and theman has not yet been able to take advantage of them.

From this point of view, we try to bring our contribution tothe research of new natural molecules economically accessible andwith an effective biological activity. We are interested in Medicagosativa, an excellent fodder plant which belongs to Fabaceae family,associated with many curative properties based on an empiricaland traditional knowledge. However, clinical studies confirmingtheir biological value are still preliminary for certain therapeuticproperties or even nonexistent for others. Our objective consistsof the study of two groups of active substances: flavonoids andsaponins extracted from leaves of Medicago sativa according to Leeet al. (1995) and Applebaum et al. (1969) protocols.

Our first goal is to determinate the yield of the two isolatedactive substances and to achieve the chemical analysis of theirfractions by thin layer chromatography (TLC). The second objec-tive is to evaluate in-vitro biological activity of twelve bacterialstrains responsible for numerous infections in humans, including

three reference strains and nine strains isolated from a hospital. Forthis purpose, the diffusion method on agar disks impregnated withplant extracts is used (Bauer and Kirby, 1966). The current bioassayis estimated by measuring the diameters of the inhibition zones ofthe bacterial growth.

The results of the physico-chemical aspect of the plant extractsindicate different yields and a diverse chemical compounds compo-sition for the both studied groups of substances. For the biologicalactivity tests show an antibacterial effect translated by a significantsensitivity of the most bacterial strains treated with flavonoids andsaponins. The degree of sensitivity varies among strains and plantextracts. However, some bacteria have shown a resistance to ourcompounds.

These results constitute a first step in the study of active sub-stances of Medicago sativa and which encourage and need toconduct additional tests to confirm the specific use of the studiedsubstances (flavonoids and saponins) to treat the bacterial infec-tions found to be sensitive to our extracts.

doi:10.1016/j.jbiotec.2010.09.794

[P-P&F.92]

AtCyclinD enhances secondary xylem differentiation and eval-uation of mitotic activity in cambium using Cyclin-GUS fusionprotein constructed from poplar

Takeo Fujii 1,∗, Kanna Sato 1, Nobuyuki Nishikubo 2, NorikoMatsui 1, Takayuki Furuichi 1, Shinya Kajita 1

1 Tokyo University of Aguriculture and Technology, Japan2 Oji paper Co. Ltd., JapanKeywords: Arabidopsis cyclinD; Vascular cambium; Secondaryxylem; Cyclin-GUS

Most of above-ground biomass is composed of secondary xylemof the wood trunk. The secondary xylem of the trunk is composedof the daughter cells produced by vascular cambium in the stem.Proliferation of woody cell is performed by long-range cell divi-sion. Most secondary xylem cells have a thickened secondary cellwall, constituting large amount of biomass storage. Therefore, theregulation of cell division in vascular cambium and differentiationinto the secondary xylem is important for biomass production. Celldivision is regulated by a lot of cell cycle regulators. In this study,we observed influence of cell cycle regulators on cell division invascular cambium in tobacco and poplar.

First, we generated transgenic tobacco and poplar that overex-pressing Arabidopsis CyclinD (AtCYCD) or AtE2F/AtDP; which havebeen reported as the positive regulators of cell cycle, and observedinfluence of the genes on vascular cambium. As a result, the bothtobacco and poplar overexpressing AtCYCD had many secondaryxylem cells than that of wild-type, and accumulated much phenoliccomponent in thickened secondary xylem cells.

Second, in order to evaluate cell division activity in vascularcambium, we constructed mitosis marker, Populus trichocarpa (Pt)CYCB1;3pro.:GUS contained Destruction box. CYCB has the regionfor ubiquitination called Destruction box, which enable to specif-ically express during mitosis phase. As a result, GUS staining wasspecifically observed in meristem region. In the transgenic tobaccoand poplar overexpressing AtCYCD, much stronger GUS stainingwas observed along with vascular cambium than that of wild-type,suggesting enhanced cell division in vascular cambium.

In conclusion, we succeeded to get novel information aboutcell division in vascular cambium with cell cycle regulators.The result would expect to bring new possibility for the pro-