Propagation of Hinoki Cypress (Chamaecyparis obtusa) Through Tissue Culture Technique as a Sustainable Method for Mass Cloning of Selected Trees

Propagation of hinoki cypress (Japanese cypress, Chamaecyparis obtusa, Cupressaceae) through adventitious bud multiplication was performed using leaf-segment explants from cutting plants of selected adult trees. Explants were successfully surface-sterilized (>90% asepsis) by agitating them in 2.5% (w/v available chlorine) sodium hypochlorite solution for 15 min and then rinsed with sterile distilled water. Explants approximately 2 cm long were cultured on plates containing medium supplemented with 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D), 20 g/L sucrose, and 7 g/L agar. The cultures were kept at 25 ± 1 °C under a 16-h photoperiod with a photon flux density of approximately 65 µmol m−2 s−1. The optimal adventitious bud multiplication (31.5 buds per explant) was obtained on a medium supplemented with 10 µM BAP in combination with 1 µM 2,4-D. Proliferated adventitious buds were elongated better on medium supplemented with 1 µM trans-zeatin. The best rooting result (86%) was achieved on a rooting medium supplemented with 1 µM 3-indolebutyric acid in combination with 0.1 µM 1-naphthaleneacetic acid. However, rooting response varied according to genotypes. Clones related to the cultivar ‘Nangouhi’ (Na18, Na14 x Isa, Na14-14, Isa x Na14, and NaS) were easier to root than those derived from the cultivar ‘ShizuokaKenZairai’ (SKZ5 and SKZ8). Regenerated plantlets did not show morphological abnormalities and showed a high survival rate after acclimatization (>90%).