FAIR-CT98-5056 Regeneration and micro-propagation of transgenic Eucalyptus globulus plants

Contacts




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Crops for Paper/Pulp : FAIR Area 1.3 - Forestry-Wood Chain : FAIR Marie Curie Research Training Grants

Objectives:

This plant biotechnology project was a continuation of a previous, the purpose of which was to use genetic engineering techniques for improving Eucalyptus globulus. E. Globulus is a tree extensively grown in southern Europe for paper pulp production. The main objective of this study was the final optimisation of an in planta procedure for transferring genes into E. globulus adult clonal material and to study the regeneration of the transformed plants.

Activities and Results:

It was found that it was possible to obtain (for the first time) stable transformation efficiency of up to 40% for the selected clone when the following conditions were established:

• Explant preculture of three weeks in the R7 medium with dark/photoperiod combination;
• Agrobacterium culture in liquid LB medium in the presence of acetosyringone and proline;
• Agrobacterium resuspension in liquid MS medium with 2,4-D, also in the presence of acetosyringone and proline;
• Transformation by wounding/infection technique of sonication 60 s followed by infiltration (five minutes)
• The use of a solid co-culture R7 medium in presence of acetosyringone.

Histochemical analysis showed that leaves, shoots and meristems expressed uidA gene at transient level, whereas mostly leaves expressed the gene 45 days after transformation. In order to be able to regenerate plants through adventitious organogenesis from the transformed tissues, 18 different media, containing higher cytokinins (BA, CPPU, TDZ) and lower auxins (NAA) concentrations, were tested initially on non-transformed leaves. From successive experiments, it was possible to establish a regeneration medium that, containing TDZ at 1 mg/ml and BAP at 2 mg/1, resulted in the lowest level of necrosis in the highest percentage of explants producing buds and the highest percentage of buds producing shoots. The new shoots were able to develop normally and grow from the new buds under the right micro-propagation conditions.

Conclusions:

The results indicated the ability of the selected material to be transformed and regenerated using the procedures developed, thus successfully satisfying the main objective of the project.

Keywords: Eucalyptus, transformation, micropropagation.

Contacts

Scientific Supervisor

• Pedro GALLEGO / Universidad de Vigo Facultad de Ciencias, Fisiologia y Biotecnologia Vegetal / SPAIN