Optimal cytokinin/auxin balance for indirect shoot organogenesis of Eucalyptus cloeziana and production of ex vitro rooted micro-cuttings

Shoot organogenesis is critical for the shortening of long breeding cycles and circumvent the barrier of cloning mature Eucalyptus cloeziana trees.It enables large-scale production of plants from transformed tissues.This study evaluates the effect ofα-naphthaleneacetic acid(NAA),thidiazuron(TDZ)and benzylaminopurine(BAP)on the organogenesis of E.cloeziana from hypocotyls and cotyledonary leaves.In the induction stage,hypocotyls and cotyledonary leaves were established in a Murashige and Skoog(MS)culture medium supplemented with NAA or TDZ.Callus tissues were cultivated in a MS culture medium containing only BAP or different concentrations of BAP/NAA in the differentiation stage.Adventitious buds were multiplied in vitro and elongated in a WPM culture medium supplemented with 0.89μM BAP and 0.05μM NAA.Cotyledonary leaves exhibited the best in vitro regeneration.The induction of adventitious buds occurred only in calluses induced from TDZ.In the differentiation stage,4.4μM BAP treatment promoted an increase of adventitious bud regeneration.Micro-cuttings from regenerated shoots were acclimatized and rooted ex vitro in mini-incubators.The results confirm the establishment of an efficient protocol for the in vitro regeneration of E.cloeziana by indirect organogenesis,providing new insights regarding cloning of this species.