S478 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

[P-P&F.19]

Heat shock induced excision of selectable marker genes in trans-genic banana by the Cre-lox site-specific recombination system

Borys Chong-Pérez 1,2,∗, Geert Angenon 2, Rafael G Kosky 1, Mar-itza Reyes 1, Luis E. Rojas 1

1 Instituto de Biotecnología de las Plantas, Universidad Central “MartaAbreu” de Las Villas. Carretera a Camajuaní km 5.5, Santa Clara, Cuba2 Laboratory of Plant Genetics, Institute for Molecular Biology andBiotechnology, Vrije Universiteit Brussel, B–1050 Brussels, BelgiumKeywords: Agrobacterium tumefaciens; auto-excision; genetictransformation; Musa

Selectable marker genes are frequently used for transgenicevent production, but after the selection process these genes are notlonger needed. Moreover, they may cause public concern and tech-nological problems. Several excision systems exist but few havebeen optimized or shown to be functional for clonally propagatedcrops. Marker free transgenic banana plants cv. Grande Naine (MusaAAA) have been obtained using a Cre/loxP auto-excision strategy.We used a binary expression vector in which the recombinasegene cre under the control of a heat shock promoter and selectablemarkers genes cassettes were placed between two loxP sites indirect orientation, while the gene of interest was inserted outsideof the loxP sites (Figure 1A). Heat shock promoters GmHSP17.6-L

Fig. 1. Outline of the marker removal strategy and PCR analysis. A, Schematicrepresentation of the original T-DNA used for transformation (HCN) and the exci-sion fragment after Cre-mediated recombination (N). B, PCR results. PCR1, primersC-3300-F/nptII-SR yield a 724 bp fragment specific for the excision allele; PCR2,primers codA-1/nptII-SR yield a 1441 bp fragment specific for the original allele.Lanes 1 to 16, DNA purified from transgenic plants submitted to heat shock; C-,untransformed plant; pGmhsp-A, plasmid control; H2O, water. M: Molecular weightmarker Gene RulerTM DNA Ladder Mix (Fermentas).

and AtHSP18.2, from soybean and Arabidopsis respectively, weretested. The results showed that a transient heat shock treatmentof primary transgenic embryos is sufficient for inducing cre andexcising the cre, hpt and codA genes. Excision efficiency, as deter-

mined by PCR (Figure 1B) and Southern hybridization, was 59.7 and44.0% for GmHSP17.6-L and AtHSP18.2 promoters, respectively.Spontaneous excision was not observed in 50 plants derived fromuntreated transgenic embryos. The system described here is simpleand might be extensively applicable for the production of marker-free transgenic plants of many crop species.

doi:10.1016/j.jbiotec.2010.09.723

[P-P&F.20]

Cloning and Characterization of Chalcone Synthase Gene fromPueraria candollei var. mirifica

Piyachat Wiriyaampaiwong 1,∗, Pornthap Thanonkeo 1, SudaratThanonkeo 2

1 Khon Kaen University, Thailand2 Mahasarakham University, ThailandKeywords: Pueraria candollei var. mirifica; Chalcone synthase;Flavonoid biosynthesis

Pueraria candollei var. mirifica is an indigenous herb found in theNorth, West and Northeastern part of Thailand. This plant synthe-sized and accumulated several biological active compounds namelyphytoestrogen such as miroestrol, deoxymiroestrol, daidzin, puer-arin, genistin, mirificin, kawakhurin, coumestrol, daidzein andgenistein. Chalcone synthase (CHS; EC 2.3.1.74), which catalyzesthe formation of chalcone from p-coumaroyl and malonyl CoAs, isthe key enzyme in flavonoid biosynthesis in plant. In this study,cloning and characterization of chalcone synthase gene (CHS)from P. candollei were carried out. Using a reverse transcription-polymerase chain reaction (RT-PCR) strategy, the CHS gene wascloned from P. candollei. The complete PcCHS coding sequencecontained 1170 bp, encoded for a polypeptide of 389 amino acidresidues with a calculated molecular mass of 42.8 kDa and pI of 5.31.Southern blot hybridization of genomic DNA fragments revealedthe presence of multiple CHS genes in the P. candollei genome.Homology analysis revealed that the deduced amino acid sequenceof PcCHS had 86-93% identity, whereas the nucleotide sequence had81-95% identity to CHS of other plants, such as P. montana, Glycinemax, Phaseolus vulgaris, Vigna unguiculata, Pisum sativum and Med-icago sativa. Expression analysis of the PcCHS gene in different planttissues by RT-PCR revealed that this gene is expressed in a tissue-specific manner. The expression of the PcCHS gene was remarkablyinduced by high temperature, UV-B and wounding treatments,suggesting that the CHS gene product may plays a crucial role in pro-tecting plant from environmental or external stresses. The resultsin this study provide useful information for further work on pro-duction of phytoestrogen, an active compound in this plant.

doi:10.1016/j.jbiotec.2010.09.724

[P-P&F.21]

Assessing genetic diversity of Tamarix spp. in three populationsin Southern Italy

S. Terzoli ∗, G. Abbruzzese, I. Beritognolo, M. Sabatti, R. Valentini,E. Kuzminsky

University of Tuscia, ItalyKeywords: Tamarix; Microsatellite; Genetic variability; Abioticstress

Tamarix plants are resistant to abiotic stresses, as they thrivein zones where drought, soil salinity, and high temperature are