Improved walnut mass micropropagation through the combined use of phloroglucinol and FeEDDHA

Abstract

Despite the socioeconomic importance of walnut trees, poor rooting and recalcitrance to in vitro culture have hampered the establishment of high-yield clonal plantations. To improve walnut micropropagation, we introduced several modifications to current methods and evaluated the effects on microshoot performance and acclimatization. Nine selected genotypes (13-year-old trees) of the commercial hybrid Juglans major 209 × J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 µM BA and 50 µM IBA. A protocol was developed that relies on the sequential use of 0.4 and 0.2 mM phloroglucinol during shoot multiplication, but not at later stages, so as to maximize shoot growth without inhibiting root formation. Moreover, substituting FeEDTA by FeEDDHA diminished chlorotic symptoms and significantly improved the rooting ability of all genotypes, with up to 90 % microshoots developing viable roots at 6.81 mg/L Fe³⁺. The addition of 83.2 mM glucose during the root expression phase was particularly efficient at promoting plant survival during acclimatization, compared to equimolar amounts of the alternative sugars sucrose and fructose. At the proposed working concentrations, the aforementioned compounds did not cause any deleterious effects on the nine genotypes studied. Microscopic analysis revealed the physical continuity between adventitious roots and stem cambial tissue. Analysis of leaf genomic DNA with eight polymorphic microsatellite markers was supportive of the clonal fidelity and genetic stability of the micropropagated material. Successful clonal plantations (over 5800 ramets) have been established by applying this protocol.