TRANSFORMATION OF A NICKEL HYPERACCUMULATING ECO-TYPE OF ALLYSUM MURALE WALDST. ET KIT. Branka Vinterhalter, Jelena Savić, Slavica Ninković, Martin Raspor, Nevena Mitić and Dragan Vinterhalter Institute for Biological Research “Siniša Stanković”, Belgrade, Serbia Balkan as a region is known for numerous Allysum species among which many are endemic. One of the reasons for such fast speciation was their ability to addapt to various types of soils including those with high content of heavy metals. Thus A. markgrafii from mountain Goc is a metal hyperaccumulator which thrives on soil rich with nickel. These plants can contain more than 2% dry weight of nickel in their tissues. Another one, A. moellendorfianum from Herzegovina is adapted to Dolomite soil types rich with Ca and Mg carbonates and fail to grow on other soils. Some non-hyperaccumulating Alyssum species like A. murale are cosmopolites growing through most of the Europe. But A. murale plants from regions in which soils are rich with heavy metals are also hyperaccumulators. These plants are considered eco-types since they do not differ from plants growing on other soil types. In our laboratory we study various Allysum species under in vitro culture conditions. Here we present a study in which we elaborated a procedure for transformation of a nickel hyperaccumulating A.murale eco-type with A. rhizogenes A4M70GUS. Experimental Plants: Shoot cultures were established from epicotyls of seedlings aseptically germinated on hormone-free MS medium. Prior to inoculation cultures were maintained on 0.1 mgl-1 kinetin which provided good multiplication and no vitrification. Bacterial strain: Agrobacterium rhizogenes strain A4M70 GUS (Tepfer and Casse Delbart 1987) contains a co integrative plasmid with a GUS construct integrated into the TL region of the pRiA4. GUS construct contains uidA sequence under the 70S promoter (enhancer-doubled 35S Ca MV promoter), followed by NOS polyadenylation sequences. Transformation: was performed by wounding shoots with a needle dipped in bacterial suspension. Shoots were further cultured 7-10 day on hormone-free medium and then transferred to medium with 500 mgl-1 Tolycar. Cultures were tested for presence of bacteria Hairy roots which appeared at the inoculation site were excised and cultured in Petri plates on hormone free MS medium GUS assay - Transformed plants were screened by GUS assay (Jefferson et al. 1987) Histological analysis standard paraffin techniques, and combined haematoxyline - GUS staining were used to locate sites of shoot regeneration on hairy roots. After 30 days 25% of the 124 inoculated explants produced hairy roots on the wounding site. Roots were excised, each was further maintained on the hormone-free medium as separate clone giving a total of 31 different clones. Hairy root clones were highly variable in their growth responses. Thus clones 3 and 6 had very fast growth and regenerated shoots spontaneously on the hormone-free medium. On the same hormone-free medium clones 2, 12, 23, 24 and 25 produced calii. Shoot regeneration in other clones required presence of cytokinins. Effect of 0 – 2.0 mgl-1 BA or 1.0-3.0 mgl-1 TDZ on shoot regeneration was investigated for all 31 hairy root clones. However only clones 7, 12, 23, 24 and 25 could regenerate shoots on cytokinin supplemented media (Tab2.). Shoot regeneration for other clones was not obtained. All regenerated shoots were short and vitrified. They were further cultured on hormone-free medium until normal growth resumed. Histological analysis of shoot regeneration revealed it to be either direct or indirect. In direct regeneration shoots developed directly from root tissues with little adjacent callus formation. In indirect regeneration shoots differentiated from callus developing on roots. Most of the meristematic centers giving rise to shoots were located on surface of explants although some were positioned deep within tissues. Combined staining with GUS and haematoxiline shows that meristematic cells comprising shoot buds manifest very strong GUS staining Hairy root induction Hairy root culture Ni- hyperaccumulation of transformed and non-transformed shoots was tested by their ability to grow on media supplemented with 1, 4, and 8 mM of NiCl2 x 6H2O. (Table 2). All media contained 0.1 mgl-1 kinetin. Both transformed and non-transformed cultures survived on media supplemented with high nickel concentrations. They affected only old leaves already present on explants which lost their green color. New shoots and their leaves, developing from axillary buds were not affected by high nickel concentrations. Growth parameters are presented separately in the table. Regenerated shoots from clones 3, 6, 12, 23 and 25 after two passages on hormone-free medium resembled plants of normal phenotype. They were further cultured on media with 0.1 mgl-1 kinetin same as non-transformed shoots used in studies on nickel hyperaccumulation. Shoot regeneration Shoots of transformed clones were characterized by good elongation and lateral shoot branching, short internodes, minute slightly curled leaves and well developed plagiotropic root system spreading over the surface of media. Thus plants which were regenerated from A. murale hairy roots cultures manifested some of the Ri syndrome phenotype characteristics. Shoots of all five putative transformed clones had a positive GUS reaction. Deep blue color appeared in all vegetative organs and tissues of transformed plantlets. Transformation was also confirmed by PCR analysis. NiCl2 x 6H2O (mM) Shoot length, (mm) Non-transform. Transformed Lateral shoots, (No.) Non-transform. Transformed 27.7 ± 1.5 21.3 ± 1.1 2.3 ± 0.2 4.2 ± 0.4 1 23.2 ± 1.4 17.0 ± 0.9 2.5 ± 0.4 3.7 ± 0.4 4 19.6 ± 0.6 17.4 ± 0.8 1.9 ± 0.2 2.8 ± 0.3 8 16.7 ± 0.6 14.5 ± 0.4 1.3 ± 0.2 2.1 ± 0.3 Transformed shoot culture Clones Shoot regeneration BA mgl-1 Hormone -free 0.1 0.2 0.5 1.0 3 Yes No 6 7 12 23 24 25 Table 2 GUS staining PCR analysis of A. rhizogenes transformed shoot cultures: lines A- 1 kb DNA ladder, lines B- positive control (A4M70GUS), lines C- negative control (non-transformed shoots), lines D-H - transformed shoots clones 3, 6, 12, 23, 25 respectively. Table 1