Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

GENETIC TRANSFORMATION OF OLIVE TREE (OLEA EUROPAEA L.) WITH OSMOTIN GENE AND IN SITU PROTEIN LOCALISATION IN THE TRANSGENIC TISSUES

D’ANGELI S.**, GUTIERREZ-PESCE P.*, ALTAMURA M. M.**., BIASI R.*, RUGGIERO B.*, MUGANU M.*, BRESSAN R.***, RUGINI E.*

* Dip. Produzione Vegetale Università degli Studi della Tuscia, I- 01100 Viterbo

Rugini@unitus.it

** Dip. Biologia Vegetale, Università degli Studi “La Sapienza” , I-00185 Roma

*** Pardue University, West Lafayette, Indiana, USA

olive, somatic embryogenesis, osmotin, genetic transformation

Embryogenic masses of olive tree (cv Canino) obtained through the double regeneration technique using leaf petioles, coming from in vitro micropropagated vegetative shoots (Rugini e Caricato, 1995), were infected with Agrobacterium tumefaciens LBA4404 strain, with pKYLX71 plasmid, containing tobacco osmotin gene and NPTII, under the control of 35S promoter (Abat et al., 1996; Liu et al., 1994; Zhu et al., 1996). After 30 days of culture under darkness, putative transgenic somatic embryos were macroscopically selected under light on a liquid medium containing kanamicin (150mg/l). The green embryos were separately cultured to produce plants and to investigate the presence of the osmotin gene, whereas the chlorotic embryos were either wasted or used as control for the molecular analyses.

Both the transgenic and control plants were transferred into a greenhouse and successively transplanted in the field, after the permission of the “Ministero della Sanità”. After three growing seasons, the plants showed a compact phenotype with short internodes and reduced-in-size leaves, and exhibited low vigour. To check the possible acquired resistance to fungal pathogens, some transgenic plants were grown in pots under high humidity conditions together with plants affected by heavy symptom of Spilocaea oleagina (Cast). Hugh.

The in situ localisation of osmotin was performed by using a polyclonal primary antibody against osmotin jointed with a secondary antibody with alkaline phosphatase (AP) activity. The AP reaction in presence of NBT/BCIP substrate (Pharmacia) causes a purple colour into the plant tissues.

In transections of one year old stems, coming from the apical internodes of field-grown plants, osmotin labelling was observed in epidermal and subepidermal tissues and, rarely, also in the most superficial layers of the cortical parenchyma. At cellular level, the protein was mainly localised around the vacuole. The signal was also present in the phloem and, mainly, in the cambium, but to a lesser extend than in the superficial tissues. In some cases, immature deuteroxylem cells resulted also labelled.