Abstract:

Objective: In this study, we investigated variations in rooting capacity and endohormones levels of subcultured shoots during long-term successive subculture of Pinus massoniana, and analyzed the correlations between endohormones levels and rooting capacity, in order to reveal rooting mechanisms and provide a theoretical basis for establishing an efficient micropropagation technique of P. massoniana. Method: The subculture buds of glm-8 genotype, derived from the 26-year-old P. massoniana 'Tongmiansong' stands, with strong rooting ability were obtained from the subculture from 2014-2018, and used as experimental materials. The rooting capacity (rooting root and root number) and endohormones levels were observed in subcultured shoots at the 1^st, 3^rd, 5^th, 8^th, 10^th, 15^th, 20^th, 30^th and 40^th subculture, respectively. The correlations between rooting capacity and endohormones levels were analyzed using linear regression, and significant differences in rooting rate, root number as well as endohormones levels among subculture times were tested based on analysis of variance. Result: 1. Subculturing times significantly affected rooting capacity of in vitro shoots in P. massoniana. For subcultured shoots of initial generations, rooting rate was 83.1% and root number was 2.4. The rooting effect of shoots was the best after subculturing of 15~20 times, reaching rooting rate of 98.7% and root number of 6.9, while the rooting ability of shoots dramatically decreased after subculturing of 40 times, when the rooting rate and root number were 65.7% and 0.5, respectively. 2. The levels of endogenous indoleacetic acid (IAA), gibberellins (GAs), abscisic acid (ABA) and zeatin riboside (ZR) significantly changed during long-term successive subculture. Among them, IAA or GAs had relatively greater correlation with the rooting capacity than ABA or ZR. Results of linear regression analysis showed that there was a significantly positive linear relationship between IAA/GAs ratio and rooting rate or root number. 3. Under the treatments of exogenous naphthaleneacetic acid (NAA), IAA and/or GA synthetic inhibitor, paclobutrazol (PAC), rooting performances were significantly different among the three subcultured buds with significant difference in rooting ability. For shoots subcultured 35~40 days, exogenous IAA improved the number of adventitious root and the optimal performance of rooting was found with the application of exogenous 4 μmol·L^-1 IAA in the rooting medium. However, PAC was obviously inhibitive for rooting of subcultured shoots. For shoots subcultured 700~800 days, NAA of low concentration (1.2 μmol·L^-1) significantly promoted adventitious rooting, while both exogenous IAA and PAC inhibited the rooting of shoots, and the inhibitive effects increased with the concentration of IAA and PAC. For shoots subcultured 1400~1600 days, IAA and PAC separately enhanced root number and rooting rate, but there was a threshold value for both IAA and PAC regarding their promotive effects on rooting. The best rooting effect was observed on the medium containing 4 μmol·L^-1 IAA+4 μmol·L^-1 PAC, and rooting rate and root number increased 49.3% and 406.7% compared with the control (NAA) treatment, respectively. Conclusion: The rooting ability of subcultured buds of Pinus massoniana is different with different subculture time, and long term subculture results in the decline of rooting ability. Endogenous IAA and GAs levels are closely related to the development of adventitious root in P. massoniana, and they regulate the occurrence and formation of adventitious roots. To achieve the efficient and stable rooting performance, we suggest that exogenous growth regulators, NAA, IAA and/or PAC, added in the medium should be chosen according to the endogenous IAA and GAs levels in micropropagated materials with different rooting capacity.

Key words: Pinus massoniana, successive subculturing, endohormone, adventitious root development, clone