Vegetative Propagation of Phytophthora cinnamomi-Tolerant Holm Oak Genotypes by Axillary Budding and Somatic Embryogenesis

Abstract

Holm oak (Quercus ilex) is one of the most widely distributed tree species in the Mediterranean basin. High mortality rates have been observed in holm oak populations in the southwest of the Iberian Peninsula as a result of oak decline syndrome. Selection and propagation of genotypes tolerant to this syndrome could aid the restoration of affected areas. In this article, we report micropropagation and conservation procedures based on axillary budding and somatic embryogenesis (SE) of holm oak plants, selected for their tolerance to Phytophthora cinnamomi—the main biotic factor responsible for oak decline. Forced shoots were obtained from potted plants of eight different genotypes, and used as stock material to establish in vitro shoot proliferation cultures. Reliable shoot proliferation was obtained in seven out the eight genotypes established in vitro, whereas multiplication rates were genotype-dependent. The highest rooting rates were obtained by culturing shoots for 24 h or 48 h on rooting induction medium containing 25 mg L−1 indole-3-butyric acid, followed by transfer to medium supplemented with 20 µM silver thiosulphate. Axillary shoot cultures can be successful conserved by cold storage for 12 months at 4 °C under dim lighting. Shoot tips, excised from axillary shoot cultures established from tolerant plants, were used as initial explants to induce SE. Somatic embryos and/or nodular embryogenic structures were obtained on induction medium with or without indole-acetic acid 4 mg L−1, in two out the three genotypes evaluated, and induction rates ranged between 2 and 4%. Plantlet recovery was 45% after two months cold stratification of somatic embryos and eight weeks of culture on germination medium. Vegetative propagation of P. cinnamomi-tolerant Q. ilex trees is a valuable milestone towards the restoration of disease-affected areas.

Micropropagation, Characterization, and Conservation of Phytophthora cinnamomi-Tolerant Holm Oak Mature Trees

“…However, no significant differences were observed in the establishment of either type of material in holm oak genotype VA11. This finding is further proof of the extreme recalcitrance of this plant species to vegetative propagation, as has also been reported regarding the in vitro establishment of juvenile plants [30]. Important differences in the success of in vitro establishment were observed between the trees from the two geographical regions considered, since trees from the Mediterranean region yielded better results than those from the southwestern region.…”

“…Rooting is a crucial step in the success of micropropagation by axillary budding [58]. However, holm oak is considered a difficult-to-root species [59], and the rooting rates reported for this species are low, even in material of juvenile origin [30]. Here, rooting was achieved in the three tested clones, but acceptable rooting values were obtained only in genotype VA5.…”

“…Axillary budding enables conservation of selected genotypes but is expensive and has the additional risk of contamination and somaclonal variation [29]. To prevent these risks, the period between each subculture event could be increased (thus reducing costs) by applying slow growth methods [30,31] or by completely inhibiting growth (cryobiotechnology) [29,32]. To date, very few studies have investigated preservation procedures in holm oak, such as cold storage to preserve established axillary shoots from juvenile plants [30], while in [33], the authors reported the cryopreservation of holm oak somatic embryos in a procedure that did not enable long-term preservation.…”

Recent Advances in Adventitious Root Formation in Chestnut

“…Moreover, other parameters, like mean root number can be affected by the auxin treatment [ 33 , 91 ]. The genotype effect on rooting performance has been previously documented in other difficult-to-root species like walnut [ 103 ], oak [ 104 , 105 ], American beech [ 106 ] and holm oak [ 107 ]. In conclusion, besides other factors, genotype is critical in the formation of ARs in woody species.…”

Application of Tissue Culture in Plant Reproduction

“…Regarding the application of SE to improve tolerance/resistance to biotic stress, Martínez et al [5] used SE to propagate holm oak plantlets selected for their tolerance to Phytophthora cinnamomi. Initially, axillary shoot cultures were established from tolerant plants, and these cultures were used to provide the shoot tips used as the initial explants for SE induction.…”