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

Abstract

A method for a high frequency and direct in vitro bud regeneration of a woody species, the trifoliate orange ( Poncirus trifoliata L. Raf), was designed. Transverse thin cell layer (tTCL) explants excised from the stem internodes of 1-year-old young plants of P. trifoliata regenerated bud in vitro on a medium containing 6-benzylaminopurine (BAP 1-50 μM) and N-phenyl-N'-1,2,3-thidiazol-5-ylurea (thidiazuron, TDZ) (0.1–10 μM). The optimal concentrations for bud induction were 25 μM BAP and 1 μM TDZ leading to 87 and 72 % of responsive tTCLs and 24 and 15 buds per tTCL, respectively. A higher percentage of responsive tTCLs and a higher frequency of bud regeneration were obtained with BAP and TDZ combined. With a combination of 10 μM BAP and 1 μM TDZ, 90 % of responsive tTCLs forming 37 buds per tTCL were obtained. Shoot elongation occurred after a transfer onto a medium containing 1 μM GA 3. Rooting of individual shoot was induced using 5 μM NAA. One hundred per cent of rooted shoots developed normally after transfer to the greenhouse; no phenotype variation was observed. High numbers of regenerated viable plants can be produced directly without callus formation from tTCL after 9 weeks of culture.