OPTIMISATION OF AN INTEGRATED BOMBARDMENT AND AGROBACTERIUM INFILTRATION BASED TRANSFORMATION SYSTEM FOR LENTIL

Celikkol, Ufuk
M.S., Department of Biotechnology
Supervisor: Prof. Dr. Huseyin Avni Oktem
Co-Supervisor: Dr. Ismail Kusmenoglu
January 2002

In this study, the effect of microwounding by particle bombardment and the coupling of vacuum infiltration to Agrobacterium-mediated transformation, on the efficiency of transient GUS expression of cotyledonary node tissues were investigated. In each experimental set with any Agrobacterium/plasmid couple, five different treatments were applied to compare the efficiency of integrated bombardment-infiltration system to reference treatments.

Throughout the study also the efficiencies of four strains, LBA4404, EHA105, C58C1, KYRT1 and two binary plasmids, pGUSINT and pTJK136 on transient GUS expression of cotyledonary node explants were compared.

Although differ upon inoculation of different strain/binary plasmid couples, overall transient expression appeared to be reduced when tissues were microwounded. Furthermore, no significant (p>0.05) effect of the vacuum infiltration was observed. Therefore, efficiency of another wounding method, mechanical injury (poking by a needle) of the cotyledonary petiole axil region was investigated.

Usage of the most efficient strain/plasmid couple, which is determined to be KYRT1:pTJK136, and mechanical wounding of the cotyledonary petiole axil region of the explants prior to Agrobacterium inoculation produced a number of transformed shoots, which show stable GUS expression in a chimeric manner. Highly regenerable nature of the cotyledonary node explants and application of the above mentioned transformation protocol seem to increase the probability of obtaining transgenic plants in lentil.

Key words: Lentil, Cotyledonary Node, Agrobacterium tumefaciens, Microprojectile Bombardment, Vacuum Infiltration, GUS