High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell layer method

A method for a high frequency and direct in vitro bud regeneration of a woo dy species, the trifoliate orange (Poncirus trifoliata L. Raf), was designe d. Transverse thin cell layer (tTCL) explants excised from the stem interno des of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 mu M) and N-phenyl-N'-1, 2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1-10 mu M). The optimal concen trations for bud induction were 25 mu M BAP and 1 mu M TDZ leading to 87 an d 72 % of responsive tTCLs and 21 and 15 buds per tTCL, respectively. A hig her percentage of responsive fTCLs and a higher frequency of bud regenerati on were obtained with BAP and TDZ combined. With a combination of 10 mu M B AP and 1 mu M TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were o btained. Shoot elongation occurred after a transfer onto a medium containin g 1 mu M GA(3). Rooting of individual shoot was induced using 5 mu M NAA. O ne hundred per cent of rooted shoots developed normally after transfer to t he greenhouse; no phenotype variation was observed. High numbers of regener ated viable plants can be produced directly without callus formation from t TCL after 9 weeks of culture. (C) 1999 Academie des sciences/Editions scien tifiques et medicales Elsevier SAS.