Improved secondary embryo production in Quercus alba and Q. rubra by activated charcoal, silver thiosulphate and sucrose. Influence of embryogenic explant used for subculture
Plant Cell Tissue and Organ Culture
Martínez MT, Vieitez AM,Corredoira E*
Instituto de Investigaciones Agrobiológicas de Galicia (CSIC), Apartado 122, 15705, Santiago de Compostela, Spain
*Author for correspondence: Elena Corredoira; e-mail:
Online resource 1: Induction of somatic embryogenesis in Quercus rubra
We define, for the first time, a reproducible protocol for induction of somatic embryogenesis (SE) and maintenance of embryogenic lines from tissues derived of Q. rubra trees. Axillary shoot proliferation cultures were used as the source of explants for initiationof somatic embryos. Stock shoot cultures were establishedin vitro from nodal explants excised from forced shoots in branch segments of three 7-year-old Q. rubra trees designated ROQ-8, ROQ-10, and ROQ-11, following the methodology described by Vieitez et al. (2009).
Induction of SE was based on the procedure reported to initiate embryogenic systemsinQ. alba (Corredoira et al. 2012) and Q. bicolor (Mallón et al. 2013). In brief,shoot apex explants (1.5–2.0 mm long, comprising the apical meristem and 2–3 pairs of leaf primordia) and leaf explants (the two most apical expanding leaves below the shoot apex) were excised from stock shoot cultures andsubjected to asequence of three-step culture method. The three step procedure involving successive culture of explants in induction medium (M1) consisting of MS mineral salts and vitamins, 500 mg/L casein hydrolysate, 21.48 µM NAA and 2.22 µM BA. After 8 weeks of culture, the explants were transferred to fresh medium of the same composition except that NAA and BA were reduced to 0.54 µM and 0.44 µM, respectively (M2 medium) for a further 4 weeks, and transfer of explants into plant growth regulator-free medium (expression medium M3) for another 8 weeks (i.e. in total, for 20 weeks after the start of culture). Ten shoot apices and ten leaf explants were placed in 90-mm Petri dishes containing 25 ml of medium. For each genotype and explant type, at least 60–70 explants were used and the experiments were repeated three times.
Embryogenic response was achieved for all three genotypes with similar induction frequencies between genotypes (see Fig. a-d). The percentage of leaf explants producing SE were 2.3%, 3.3% and 2.0% for ROQ-8, ROQ-10 and ROQ-11, respectively. Similar responses were obtained in ROQ-8 (2.3%) and ROQ-10 (2.2%) shoot tip explants, while no somatic embryos were initiated in ROQ-11 shoot tips. Somatic embryos were isolated from original explants and clonal embryogenic lines were established and maintained by repetitive embryogenesis.
Figure a–d . Induction of somatic embryogenesis in explants from stock shoot cultures of Q. rubra. Somatic embryos initiated from leaf explants of ROQ-10 (a, b) and ROQ-8 (c) genotypes. d Somatic embryos generated from a shoot apex explant of ROQ-10 genotype.