Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and Agrobacterium-mediated transformation of leaf tissues Cesar Petri 1 , Nuria Alburquerque 1 , Olaya Pe´rez-Tornero 2 & Lorenzo Burgos 1, * 1 Departamento de Mejora y Patologı´a Vegetal, CEBAS-CSIC, Aptd. Correos 164, 30.100-Murcia Spain; 2 Vitrotech Biotecnologı´a Vegetal, Crta. Nacional 340, Km 631, 7 Finca Ascoico, 30.840-Alhama, Murcia Spain (*request for offprints; Fax: +34 968396213; E-mail: burgos@cebas.csic.es) Received 23 December 2003; accepted in revised form 30 November 2004 Key words: adventitious regeneration, Agrobacterium-mediated transformation, auxins, polyamines, Prunus armeniaca Abstract The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2 mM in combination with 0.5 lM aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same con- centration but in combination with 60 lM silver thiosulphate and four-day pulses with 9 lM 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots. Abbreviations: AS – acetosyringone; AVG – aminoethoxyvinylglycine; DKW – Driver and Kuniyuki (1984); GFP – green fluorescent protein; QL – Quoirin and Lepoivre (1977); NAA – naphthaleneacetic acid; Put – putrescine; Spd – spermidine; Spm – spermine; STS – silver thiosulphate; TDZ – thidiazuron; 2, 4-D – 2, 4 dichlorophenoxy–acetic acid Introduction The lack of efficient adventitious regeneration systems is the major limiting factor preventing the development of gene transfer technologies for perennial crops. Fruit trees are among the most recalcitrant in producing adventitious shoots. The use of ethylene inhibitors to enhance shoot regeneration has been described in some species and an efficient adventitious regeneration procedure from adult apricot leaves has been developed, which include the use of the ethylene inhibitors STS or AVG (Burgos and Alburquerque, 2003). Polyamines are low molecular weight organic cations implicated in various physiological and developmental processes in bacteria, animals and plants. The diamine Put is the precursor of the triamine Spd and the tetramine Spm. In plants, Put, Spd and Spm are present in amounts Plant Cell, Tissue and Organ Culture (2005) 82: 105–111 Ó Springer 2005