an optimized and rapid dna extraction method from leaves of grapevine suitable for pcr-dgge based...

S486 Special Abstracts / Journal of Biotechnology 150S (2010) S1–S576

represents. The scaling up of hairy root cultures is an interestingstrategy for the pharmaceutical production of these compoundsinstead of the isolation from plants what could be affected by exter-nal factors such as temperature or pathogens. The aim of this workwas to study the anisodamine and scopolamine production by B.candida hairy roots immobilized in a plastic mesh submerged intoa 1.5 l stirred tank as an alternative production system. A volumeof 1.2 L of Gamborg B5/2 culture medium was inoculated with10 g FW of 20-days-old hairy roots. The culture was aerated bya stainless steel sparger located below the turbine by an airflowrate of 0.5 vvm. The alkaloids were analyzed and quantified byHPLC with UV detection. It was found that bioreactor cultures pro-duced an increased biomass and alkaloids concentration comparedto the processes carried out in Erlenmeyer flasks. The maximumconcentration of anisodamine was 10.05 ± 0.76 mg/g DW being thepredominant alkaloid at the harvest day (20 d). The results obtainedare promissory for the development of an anisodamine productionsystem.

doi:10.1016/j.jbiotec.2010.09.742

[P-P&F.39]

Arabidopsis thaliana overexpressing Zea mays glutathione S-transferase: modelling an effective phytoremediation

A Bittsánszky 1,∗, G Gyulai 2, T Kömives 1

1 Hungarian Academy of Sciences Plant Protection Instiute, Hungary2 St. Stephanus University, Institute of Genetics and Biotechnology,HungaryKeywords: Arabidopsis thaliana; phytoremediation; glutathione S-transferase

Genetic engineering is a powerful tool to investigate plantmetabolic pathways. Overexpression or silencing specific geneshelps to clarify their roles under different conditions. Understand-ing the biochemical processes contributing to the detoxificationof xenobiotics in plants will greatly help in improving their phy-toremediation potential. Glutathione S-transferases (GSTs) are alarge and diverse family of detoxifying enzymes that catalyze theconjugation of the glutathione tripeptide with a wide variety ofsubstrates. Detoxification is a key step in phytoremediation oforganic pollutants. Therefore the role and activity of GST enzymescontribute greatly for the effectiveness of decontamination. Zeamays gst27 (Accession # X79515; phi class) is a well characterizedglutathione S-transferase gene which is highly inducible by elec-trophile xenobiotics such as chloroacetanilides. Overexpression ofsuch detoxifying enzymes in plants would be helpful to improvetheir ability to clean up polluted soil and water.

In the study presented, Arabidopsis thaliana plants were trans-formed with gst27 gene to overexpress glutathione S-transferase.Transformations were carried out with floral dip method andplants from the fourth transgenic generation were selected forhomozygosity. Transgene content, expression and copy numberwere checked with PCR, RT-PCR and quantitative PCR, respectively.Homozygous plants were tested against acetochlor, metolachlor,acifluorfen and paraquat herbicides under in vitro conditions.Strong phenotypic differences were observed when transgenicseeds were germinated on ½ MS media supplemented with meto-lachlor. Only transgenic lines formed roots while control lines grewwithout them.

Stable transgenic Arabidopsis lines are excellent tools for study-ing the detoxification of organic compounds that are widely usedin agricultural fields.

Acknowledgement

The project was supported by Hungarian Scientific ResearchFund (OTKA PD-75169)

doi:10.1016/j.jbiotec.2010.09.743

[P-P&F.40]

An Optimized and Rapid DNA Extraction Method From Leaves ofGrapevine Suitable for PCR-DGGE Based Analysis

Sandra Lo Piccolo ∗, Gaetano Conigliaro, Nicola Francesca, LucaSettanni, Santella Burruano, Giancarlo Moschetti

University of Palermo, SENFIMIZO Department, Section of Phy-topathology and Agricultural Microbiology, ItalyKeywords: CTAB; DNA extraction; PCR-DGGE; polyphenols;polysaccharides; Vitis vinifera

Molecular techniques and genetic studies require a fair amountof DNA of high quality in order to produce reliable and clear results(Li et al., 2007). In grapevine, the yield and quality of DNA can be sig-nificantly affected by secondary metabolites such as polyphenols,polysaccharides and tannins, that may be consistently abundantduring the different stages of leaf development (Iandolino et al.,2004). The development of a simple, rapid and reliable methodfor the extraction of genomic DNA from grape leaves collectedat several stages during development, as well as from healthygrape leaves and leaves infected by pathogenic microorganisms,was the main objective of this study. The protocol reported, basedon a modified cetyltrimethylammonium bromide (CTAB) extrac-tion procedure (Doyle and Doyle, 1990), allowed the rapid DNAextraction from little amounts of leaf material without employ-ment of liquid nitrogen for initial tissue grinding. The protocolincluded polyvinylpyrrolidone to bind phenolic compounds, ˇ-mercaptoethanol to inhibit the oxidation of polyphenols, and ahigh concentration of NaCl (2.5 M) to increase the solubility ofpolysaccharides, thus, reducing their co-precipitation with DNA.Final DNA solution did not contain polysaccharides, polyphenolsand other major contaminants. The quality of the DNA extractedwith the above protocol was first evaluated for its accessibility forpolymerase chain reaction (PCR); genomic DNA from healthy andPlasmopara viticola infected leaves of Vitis vinifera was success-fully amplified by traditional PCR. Subsequently, the same DNAsolution was used for amplification with a primer pair specificfor PCR-denaturing gradient gel electrophoresis (DGGE), contain-ing a GC-clamp at the 5′-end of the forward oligonucleotide. PCRproduced amplicons with the expected molecular size (200 bp)suitable to be analyzed by means of a denaturing containing poly-acrilamide gel for the bacterial ecology composition.

References

Doyle, J.J., Doyle, J.L., 1990. Isolation of plant DNA from fresh tissue. Focus 12, 13–15.Iandolino, A.B., Goes da Silva, F., Lim, H., Choi, H., Williams, L.E., Cook, D.R., 2004.

High-Quality RNA, cDNA, and Derived EST Libraries From Grapevine (Vitisvinifera L.). Plant Molecular Biology Reporter 22, 269–278.

Li, J.T., Yang, J., Chen, D.C., Zhang, X.L., Tang, Z.S., 2007. An optimized mini-preparation method to obtain high-quality genomic DNA from mature leavesof sunflower. Genetics and Molecular Research 6 (4), 1064–1071.

doi:10.1016/j.jbiotec.2010.09.744