Root formation in micropropagated shoots of Sequoia sempervirens using Agrobacterium

Abstract

Improved rooting was demonstrated in coast redwood ( Sequoia sempervirens (D. Don.) Endl.) using an Agrobacterium-mediated gene transfer system. Microcloned shoot-tips of redwood (clone CA 3) were inoculated with Agrobacterium rhizogenes wild strain 8196 or with the pRi A4 transconjugant strain of Agrobacterium tumefaciens (C58 chromosomal background) at the basal-cut end. Roots were formed in 58–69% of explants, but without a hairy-root phenotype. Rooting frequency and the number of adventitious roots on inoculated shoot-tips were slightly moderated by the bacterial strain, duration of inoculation and explant support system. Growth of excised roots was very slow on hormone-free medium and opine production could not be confirmed. Callus cultures derived from putative transformed roots were established on medium supplemented with 1.13 μM 2,4-D and 2.22 μM BA. PCR analysis of callus tissues DNA confirmed that integrative transformation had occurred in Sequoia adventitious roots generated after infection with Agrobacterium strains 8196 or A4. This is the first report of transgenic Sequoia tissue using A. rhizogenes-mediated gene transfer.