Root formation from transgenic calli of Ginkgo biloba.

Abstract

The objective of this study was to produce Ginkgo biloba L. hairy roots for future investigations into the feasibility of producing terpenoids in differentiated cell cultures. Zygotic embryos of G. biloba were inoculated with the wild agropine-type Agrobacterium rhizogenes strain A4. Three months after bacterial infection, primordia-like nodular structures formed at the root wound sites and developed into tiny calli. Calli cultivated on hormone-free Lloyd and McCown (1980) solid medium were transferred onto Murashige and Skoog (1962) solid medium, and grew rapidly for the first few months. As browning appeared and growth slowed, calli were transferred onto Ball (1959) or White (1954) solid media. These calli became nodular with several nodules from which roots, displaying characteristic features of hairy roots, developed. Transgenic calli cultivated in agitated, hormone-free liquid media led to the formation of root meristems and root tips by a cyclic development process. Stable integration of the rolA, rolB and rolC genes into calli, mature roots and root meristem and root tip mixtures was confirmed by polymerase chain reaction (PCR). The absence of a 437 bp amplificate corresponding to the virD1 gene confirmed that there was no bacterial contamination of G. biloba tissues. The reverse transcription-PCR method was used to verify the expression of rolA and rolC genes in mature roots. Expression of rolA, rolB and rolC in root meristems and root tips was confirmed by 3' RACE-PCR analysis, which excluded amplification of possible rol gene transcripts produced by residual bacteria. This paper shows, for the first time, the feasibility of developing G. biloba roots from transformed calli.