Seedling preconditioning in thidiazuron enhances axillary shoot proliferation and recovery of transgenic cowpea plants

Abstract

Lack of competence of seedling explants for efficient shoot proliferation in recalcitrant grain legume cowpea restricts its genetic manipulation for crop improvement. This study aimed at establishing a protocol to increase the shoot proliferation efficiency during the regeneration of transgenic cowpea plants. Here, we describe how seedling preconditioning in thidiazuron (TDZ) could stimulate the transformation process (by 3.5-fold), shoot proliferation potential of cotyledonary node (by a factor of fourfold) and accelerate the transgenic shoot regeneration. We investigated the effect of TDZ and 6-benzyladenine (BA) at high dose (5–20 μM) in the induction phase of regeneration by preconditioning seedlings for different durations (2–6 days) with the aim of improving shoot proliferation competence from cultured explants. Cotyledonary node explants from preconditioned seedlings were cultured on MSB5 medium supplemented with 5 μM BA and 0.5 μM kinetin for 4 weeks. Best response in terms of maximum shoot proliferation (7.1 shoots per explants), and greatest shoot length (2.6 cm) were obtained with explants derived from seedlings preconditioned in 10 μM TDZ for 4 days. This enhanced shoot proliferation ability was maintained through three subsequent 4-week long regeneration passages. On comparison of the transformation rate in absence and presence of seedling preconditioning (in 10 μM TDZ for 4 days), a significant enhancement from 0.6 to 2.1% was observed. The promotive effect of seedling preconditioning had a direct beneficial effect on transgenic plant recovery time leading to a reduction of more than 2 weeks. The protocol was found applicable to seven cowpea genotypes.