Agrobacterium rhizogenes Mediated Hairy Root Induction in Parasponia andersonii Planch

Abstract

Background and Objective: A comparative analysis of a unique Parasponia-rhizobium symbiotic system can help to define strategies for transferring nitrogen-fixing ability to non-legume crops. Hairy-root cultures and transgenic plants of Parasponia andersonii (P. andersonii) containing various target genes regenerated from them may become one of the model systems for the investigation of Parasponia-rhizobium symbiosis. Therefore, the purpose of this work was to generate Parasponia hairy roots carrying lectin pea psl gene and regenerate transgenic shoots from these roots. Materials and Methods: Stem segments of P. andersonii were transformed using Agrobacterium rhizogenes (A. rhizogenes) strain A4 containing the binary vector pCambia 1301, which is carrying the GUS (β-glucuronidase) reporter gene with catalase intron, a selective hpt gene (hygromycin) and pea lectin psl gene. Agrobacterium-mediated transformation was carried out using silicon carbide whiskers. Hairy roots cultures co-transformed with rol -genes and T-DNA of pCambia 1301 were selected using histochemical GUS and PCR analyze these roots. Student’s t-test using the GraphPad Software(2.03) was also used to analyze the data. Results: Parasponia andersonii hairy roots showed vigorous growth and abundant lateral branching in Lloyd and McCown Woody Plant Medium (WPM) without phytohormones. Spontaneous shoot regeneration in cultures of co-transformed hairy roots growing on hormone-free medium was observed with an extremely low frequency, under both light and dark conditions. The frequency of shoot regeneration increased with the addition of thidiazuron in the medium. Conclusion: Silicon carbide whiskers increase the effectiveness of Agrobacterium-mediated transformation of Parasponia. The P. andersonii hairy roots expressing the pea psl gene can be used as a model system for studying the molecular bases of Parasponia-rhizobium symbiosis.