Eucalyptus nitens plant regeneration from seedling explants through direct adventitious shoot bud formation

Abstract

A protocol for true-to-type plantlets production by direct adventitious bud proliferation from Eucalyptus nitens hypocotyl segments was developed. Hypocotyls, cotyledons, and expanded leaves of Eucalyptus nitens were cultured in Murashige and Skoog (MS) medium containing naphthalene acetic acid or indole-3-acetic acid (IAA) and 6-benzyl adenine (BA). The most effective treatment for shoot bud formation, 0.5 μM IAA and 2.5 μM BA, was used in additional experiments. After 30 days of culture, the highest rate of regeneration (40 ± 5.8%) and the maximum number of buds differentiated per hypocotyl explant (10.3 ± 4) were obtained when explants were subjected to darkness for the first 10 days and then transferred to light-emitting diode lights mimicking daylight for 20 days of incubation. Histological examination confirmed direct shoot regeneration of E. nitens. For shoot growth, half-strength MS plus 0.09 M sucrose under forced ventilation yielded elongated shoots after 1 month. After 12 weeks from the onset of an experiment, 40 ± 11.5% of elongated shoots produced 5.3 ± 0.8 roots after pretreatment with indole-3-butyric acid aqueous solution and culture on basal medium without plant growth regulators under a temporary immersion system. Inter simple sequence repeat (ISSR) marker analysis revealed the genetic uniformity among the in vitro raised plants, demonstrating the reliability of the procedure.