Shoot regeneration process and optimization of Agrobacterium-mediated transformation in Sinningia speciosa

Abstract

The florist’s Gloxinia, Sinningia speciosa, which bears considerable flower trait variations, is an emerging model plants to study floral traits development. However, the investigation of the genetic information linking these floral traits is limited due to a lack of a reliable and efficient transformation system for functional studies. This study aims to optimize a stable genetic transformation system for S. speciosa. Detailed regeneration process and tissue culture parameters are also elucidated. The results show that the plant regeneration, initiated from a single perivascular parenchyma cell, can be induced from leaf and petiole explants in the presence of 1 mg/mL 6-benzylaminopurine (BA) and 0.1 mg/mL naphthalene-acetic acid (NAA) through embryogenesis. In the presence of 0.1 mg/mL NAA only, the adventitious roots form prior to the re-differentiation of shoot tissues in leaf explants. When the proximal end of the petiole is orientated upright with the distal end to the medium, it results in higher success of regeneration, suggesting that hormone supplies must follow endogenous basipetal auxin polarity. Using a glucuronidase (GUS) reporter gene construct, maximum transformation (3.13%) was obtained after a 3 day pre-culture and 5 day co-culture from cotyledons and leaves of 3-week-old seedlings inoculating Agrobacterium strain EHA105. The putative transgenic lines were validated by RT-PCR, Southern blotting and GUS activity. Our result demonstrates that young seedlings are the best material for transformation, probably because young leaves are only a few cell layers thick allowing inner perivascular cell (the origin of regeneration) to be more accessible for Agrobacterium infiltration.