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The rooting capacity of  Pinus  massoniana  is poor, especially for mature trees, and has prevented the development of clonal forestry for  P.  massoniana . In this study, we varied explant types, subculture times and exogenous hormones for plantlet regeneration and assessed shoots for rooting rate and root number for  P.  massoniana . Following five repetitive grafts, new shoots from grafts used as explant sources were rejuvenated as observed from juvenile shoot morphology and anatomy, leading to greatly enhanced plant regeneration in comparison to that of mature materials from 26-year-old  P.  massoniana  trees. The rooting capacity of subcultured shoots increased with successive subcultures, reaching a peak at 20 subcultures with 35–40 days per subculture. However, rooting performance was significantly reduced after 30 subcultures. The addition of naphthaleneacetic acid (NAA) plus indoleacetic acid in the medium improved the root number, but the combination of exogenous NAA with paclobutrazol (PBZ) increased rooting rate and root number. We thus greatly improved the rooting capacity of mature  P.  massoniana  trees by optimizing explant types (rejuvenated), subculture times (20 subcultures, 35–40 days per subculture) and addition of NAA + PBZ to the rooting medium. The conditions can be used for efficient plantlet regeneration of  P.  massoniana .

Optimization of rhizogenesis for in vitro shoot culture of Pinus massoniana Lamb.