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BackgroundAgrobacterium mediated gene transfer technology offersthe potential to introduce novel, high-value traits intoselected, elite tree genotypes. Significant progress hasbeen made in the regeneration and genetic transforma-tion of Eucalyptus trees, but transformation efficiency hasgenerally been low for eucalypts [1], especially for eliteclonal genotypes. As a preliminary step towards theregeneration of explants derived from clonal material andthe production of transgenic plants from commerciallyimportant Eucalyptus genotypes, there is a need to iden-tify genotypes exhibiting high regeneration capacity. Inthis ongoing study, we are comparing eightEucalyptusgenotypes for callus induction and shoot regenerationpotential. Browning of callus tissue and surrounding cul-ture media is a common obstacle limiting regeneration ofshoots in eucalypts. Hence, a second aim is to minimizethe oxidation of phenolic compounds released from thewounded tissue and improve shoot regeneration rates.MethodsShoot buds collected from potted ramets of eight Euca-lyptus clones (Sappi and Mondi, South Africa) wereestablished underin vitro conditionson MS [2] basalmedium containing BAP (Benzyl adenine purine). Theestablished clones consisted of four E. grandis (G1, G2,G3 and G4), oneE. grandis x E. nitens (GN1), two E.grandis x E. camaldulensis (GC1 and GC2) and one E.grandis x E. urophylla (GU1) genotype. Leaf explantswere excised from in vitro shoot cultures and culturedon MS basal medium containing BAP, NAA(Naphthalene acetic acid) and TDZ (Thidiazuron).Browning and necrosis of the callus as well as surround-ing culture media was observed in all cultures and PVP(polyvinylpyrrolidone) was therefore added to the mediato reduce oxidation.A standardised regeneration protocol was used for theoptimization of Agrobacteriummediated genetic trans-formation of selected Eucalyptus clones. The Agrobac-terium tumefaciens strain AGL1 containing pMDC162with the uidA gene (b-glucuronidase; GUS) andhpt(hygromycin phosphotransferase) gene was used fortransformation. To minimize extensive exudation ofphenolic compounds from the wounded explants, theleaves were subjected to different wounding methods.Intact leaves were excised fromin vitro shoot culturesand preconditioned for 24 hrs on callus induction med-ium followed by wounding with a surgical blade prior toAgrobacterium infection. The leaves were wounded intwo different ways: (a) removing the edges of the leaf,making 8-10 small wounds and cutting the leaf transver-sally into two equal halves and (b) making 5-6 smallwounds in the intact leaf without damaging the rest ofthe leaf. Alternatively, the leaves were wounded prior to24 h preconditioning using the first wounding method(a). The explants were immersed in a bacterial solution(A

Optimization of a plant regeneration and genetic transformation protocol for Eucalyptusclonal genotypes