1.3.7
CAN LIPID TRANSFER PROTEIN AND DEFENSIN INCREASE RESISTANCE TO PATHOGENS IN PICEA ABIES?

M ELFSTRAND1, I SABALA1, D CLAPHAM1, A LÖNNEBORG2 and S VON ARNOLD1

1Department of Forest Genetics, Swedish University of Agricultural Sciences, PO Box 7027, S-75007, Sweden; 2Norwegian Forest Research Institute (NISK), Högskoleveien 12, N-1432 Ås-NLH, Norway

Background and objectives
Heterobasidon annosum, which causes root rot, is one of the major pathogens on Picea abies. There is genetic variation for susceptibility to root rot, but to date no fully resistant genotypes have been identified. In our work we are aiming to identify and characterize genes which can decrease susceptibility to H. annosum in P. abies. To start with, we are studying if lipid transfer proteins (LTP) and defensins can increase resistance to pathogens in P. abies, as has been shown for other species [1, 2]. A cDNA clone (Pa18) coding for a putative lipid transfer protein, and another (SPI 1) coding for a defensin-like protein [3] have been isolated from P. abies. Somatic embryos of P. abies have been transformed with Pa18 and SPI 1, and we shall test if transgenic somatic embryos have an increased resistance to H. annosum. Biotests have been set up for testing resistance in somatic embryos and in small plantlets.

Results and conclusions
Pa18 and SPI1 were coupled to strong promoters in gene constructs that include the BAR gene fused to the maize ubiquitin promoter. The gene constructs were transfered to somatic embryos of P. abies using a particle inflow gun (D Clapham, I Sabala and S von Arnold, unpublished). Stably transformed cell lines are selected on media containing BASTA at a low concentration. At present we have selected 81 embryonic cell lines transformed with SPI1, as well as 23 embryogenic cell lines transformed with Pa18. The cell lines were allowed to proliferate on non-selective media for 1-2 months before transfer to maturation medium. A large number of mature somatic embryos have been recovered, and plant regeneration is under way.

We have set up two different biotests to study the change of resistance to H. annosum: co-cultivation of H. annosum; and embryogenic callus and inoculation of young plantlets with H. annosum. We now aim to test resistance in transgenic cell lines using these biotests.

References
1. Garcia-Olmedo F, Molina A, Segura A, Moreno M, 1995. Trends in Microbiology 3, 72-74.
2. Broekaert WF, Terras FRG, Cammue BPA, Osborn RW, 1995. Plant Physiology 108, 1353-1358.
3.Sharma P, Lönneborg A, 1996. Plant Molecular Biology 31, 707-712.