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

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 1.24

TRANSFORMATION OF APPLE ROOTSTOCK WITH A Natural Plant-Defence GENE AGAINST MELOLONTHA MELOLONTA

CRICRI’ M.*, BASSO B.**, SALA F.*

*) Dipartimento di Biologia, Università di Milano

**) CNR, Istituto di Biofisica / Dipartimento di Biologia, Università di Milano

Melolontha, apple rootstock, plant transformation, insecticidal genes

Apple crops in Valle d’Aosta, Italy, are heavily endangered by Melolontha melolontha.

The larvae of this Coleopteran pest live in the soil and feed on roots. Apple rootstock are a favourite food for the insect.

Different approaches are being experimented to face the problem. These include: (a) manual removal of the larvae, (b) the use of nets covering the soil, in order to prevent butterfly disclosure, (c) the use of insecticides, (d) the use of biological competitors, such as fungi (Beauveria brongniartii, B. bassiana), bacteria (Bacillus popilliae, B. thuringiensis) or nematodes (Heterohabditis bacteriophora, H. megidis, Steinernema glaseri). Some of these approaches are promising but, for the time being costly or limited to specific environments.

In this uncertain situation, it was decided that a biotechnological approach should be considered. The experimental plan was essentially based on the identification of a gene producing an insecticidal protein active on M. melolontha. The integration of this gene in the genome of the apple rootstock should provide resistance to the larvae attack, while the grafted apple tree will not be genetically modified and thus will not produce the foreign protein.

The experimental development of the research had to face different problems: selection of an appropriate insecticidal gene, its integration in a gene construct with appropriate expression signals, its transfer to the genome of rootstock cells and the differentiation of rootstock plants from the transformed material.

As for the first point, we have been considering genes for Bacillus thuringiensis toxins, insect kitinases and proteinase inhibitors. In the case of B.t. toxin genes, we have tested the effect of 34 B.t. insecticidal principles kindly supplied by Novartis (Basel, Switzerland). However, when added to the diet of larvae grown in the laboratory, none of the principles showed insecticidal activity. It was concluded that these principles, selected by Novartis for their activity on Lepidoptera, are not effective on M. melolontha. The attention has thus been diverted to proteinase inhibitors. As a preliminary step, proteins have been extracted from the gut of second-instar larvae and analysed to verify the features of the insect proteinase(s). The results have been used to select a gene coding for an appropriate proteinase inhibitor; an Arabidopsis thaliana gene (Atcys) was chosen, that had already proved to be active in the defence of poplar trees, against the widespread leaf beetle Chrysomela populi. The Atcys gene, coding for a cysteine proteinase inhibitor, was kindly supplied by M. Delledonne, University of Piacenza.

In order to set up the methodology for gene transfer and plant regeneration, apple rootstocks (clone M9B) have been micropropagated in vitro. Transient expression of the selectable GFP (Green Fluorescent Protein) gene was obtained in young leaves following particle bombardment with an appropriate gene construct. Regeneration of apple-rootstock shoots was optimised for the M9B clone, while technical problems concerning stable transformation are now being faced taking advantage of experience in other plants.