Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean

Muthukrishnan Arun; Arunachalam Chinnathambi; Kondeti Subramanyam; Sivabalan Karthik; Ganeshan Sivanandhan; Jeevaraj Theboral; Sulaiman Ali Alharbi; Chang Kil Kim; Andy Ganapathi

doi:10.1007/s13205-016-0448-0

Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean

3 Biotech. 2016 Dec;6(2):148. doi: 10.1007/s13205-016-0448-0. Epub 2016 Jun 28.

Authors

Affiliations

¹ Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India.
² Department of Horticultural Sciences, Kyungpook National University, Daegu, 702-701, South Korea.
³ Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, 11451, Kingdom of Saudi Arabia.
⁴ Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure links 653, 9000, Ghent, Belgium.
⁵ Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, Tamil Nadu, 620024, India. aganapathi2003@bdu.ac.in.
⁶ Bharathiar University, Coimbatore, Tamil Nadu, 641046, India. aganapathi2003@bdu.ac.in.

Abstract

The present work demonstrates the participation of polyamines (PAs) to improve direct regeneration and Agrobacterium-mediated transformation in soybean half-seeds. The inclusion of PAs to culture medium along with optimal plant growth regulators (PGRs) enhanced shoot induction [98.3 %; 4.44 µM N⁶-benzyladenine (BA) and 103.27 µM spermidine] and elongation [90.0 %; 1.45 µM gibberellic acid (GA₃) and 49.42 µM spermine]. The polyamine putrescine (62.08 µM) alone greatly enriched root induction (96.3 %). The influence of PAs on transformed plant production was assessed by comparing optimized protocol (comprising PAs and PGRs) with a regeneration system involving only PGRs. Plant transformation was performed in half-seeds of cultivar DS 97-12 using strain EHA105 harboring pCAMBIA1301. Transgene expression and integration was confirmed by GUS staining, PCR, and Southern hybridization. The transformed explants/materials successively cultured on co-cultivation (BA and spermidine), shoot induction (BA and spermidine), shoot elongation (GA₃ and spermine), and rooting medium (putrescine) showed enhanced transformation efficiency (29.3 %) compared with its counterparts (14.6 %) with respective PGR alone [BA, GA₃, or indole-3-butyric acid (IBA)]. Overall findings of the study suggest that involvement of PAs improved T-DNA transfer during co-cultivation, and delivered most suitable condition for efficient regeneration/survival, which led to enhanced transformation efficiency in soybean.

Keywords: Half-seed; Putrescine; Soybean; Spermidine; Spermine.