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 9 J. regia were cultured in vitro on DKW-C medium supplemented with 4.4 lM BA and 50 lM 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 Fe 3? . 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.

Testing of different iron sources and concentrations on shoot multiplication of blackberry (Rubus fruticosus L.)

“…Except for cultivar 'Willamette', increasing iron concentration above MS level significantly decreased the number of shoots in all other cultivars similarly like double concentration of both iron sources in our study. Similar results were found also by LICEA-MORENO et al (2015) who focused on walnut (Juglans major) micropropagation. Increasing FeEDTA levels up to 1.5 times (10.21 mg l -1 Fe 2+ ) did not improve microshoot performance.…”

Analysis of nutrient deficiencies affecting in vitro growth and development of Eucalyptus dunnii Maiden

“…1d, f, g, h, i). Fe deficiency generates internerval chlorosis in young leaves in a wide variety of crops (Bergmann 1992;Broadley et al 2012); these symptoms were observed in vitro in Corylus avellana (Al Kai et al 1984), Passiflora edulis (Monteiro et al 2000), Rosa hibrida (Van Der Salm et al 1994), E. globulus microcuttings (Schwambach et al 2005), Juglans major 209 9 J. regia (Licea-Moreno et al 2015) and E. dunnii cultures (Oberschelp and Gonçalves 2016). Moreover, organogenic callus formation was observed under Fe deprivation (Fig.…”

Unraveling Factors Affecting Micropropagation of Four Persian Walnut Varieties

“…Undoubtedly, only tissue culture would offer the possibility to produce high volumes of certified materials in short time periods, maintaining the genetic identity of propagated clones. Although several authors have published micropropagation protocols for various genotypes [10][11][12][13][14][15][16][17], walnuts are considered recalcitrant to tissue culture, becoming difficult the in vitro propagation of the newly realized genotypes. Additionally, the low reproducibility of micropropagation protocols is another critical factor that makes the reproduction of some varieties a more complex process; forcing to perform their adaptation on a genotype-to-genotype base, and laboratory-to-laboratory as well.…”