Micropropagation of holm oak by axillary budding and somatic embryogenesis

Autor: Martínez Santiago, María Teresa
Přispěvatelé: Vieitez Martín, Ana Mª, Corredoira Castro, Mª Elena, Universidade de Santiago de Compostela. Escola de Doutoramento Internacional (EDIUS), Universidade de Santiago de Compostela. Programa de Doutoramento en Avances en Bioloxía Microbiana e Parasitaria, Viéitez Martín, Ana María, Estevez Toranzo, Alicia, Martínez, Santiago [0000-0002-7416-875X], Martínez, Santiago
Rok vydání: 2021
Předmět:
Zdroj: Minerva. Repositorio Institucional de la Universidad de Santiago de Compostela
Universidad de las Islas Baleares
Digital.CSIC. Repositorio Institucional del CSIC
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Popis: The sclerophyllous oak Quercus ilex L. (holm oak) is widely distributed throughout the Mediterranean basin and is one of the predominant oak species in the “dehesa” ecosystem (a Spanish agroforestry system of high natural and cultural value). In the last few decades, several factors have contributed to the drastic reduction in holm oak populations in the Iberian Peninsula, including the lack of natural regeneration, ageing of the trees, abandonment of silvicultural practices and, in particular, episodes of high mortality caused by the oak decline syndrome or “la seca”, a disease of complex aetiology that is mainly caused by the oomycete Phytophthora cinnamomi. The recommended measures for reducing and controlling the risk of spread of the disease in areas where the presence of P. cinnamomi has been confirmed include management actions and chemical control. However, to date, none of these methods has proved effective for controlling the disease. An alternative could be the repopulation of the affected areas with disease-resistant/tolerant genotypes, but genetic improvement by conventional breeding programmes to yield resistant or tolerant genotypes is time-consuming, as the reproductive cycle must be completed. Vegetative propagation of selected genotypes by micropropagation techniques is a possible option instead of traditional breeding programmes. Hence, the main objective of the present research was to investigate the use of axillary budding and somatic embryogenesis (SE) processes to propagate holm oak resistant or tolerant genotypes. For axillary budding, juvenile plants (4–7 years) selected for their tolerance to P. cinnamomi and crown branch segments from trees (30–100 years old) were force-flushed, and the new shoots were used as a source of explants to initiate cultures. The stabilization process of shoot cultures was similar between both origins, being achieved in six of the eight adult and seven of the eight juvenile genotypes evaluated. Both shoot establishment and the proliferation processes were more influenced by the genotype than the origin of the material. Efficient shoot proliferation was attained by cultivating explants in a 6-week-long culture cycle with transfer every two weeks to fresh proliferation medium consisting of Lloyd and McCown medium supplemented with 6-benzyl adenine (BA), zeatin, 30 g/L of sucrose and 8 g/L of Sigma agar. The addition of 20 µM silver thiosulphate (STS) to the shoot proliferation medium had a significantly positive effect on shoot development by reducing shoot tip necrosis and early leaf senescence. The rooting step was affected by the age of the donor plant, although the effect of genotype can be not discarded. In mature material, in vitro rooting was obtained at relatively low rates (0–19%) by using Murashige and Skoog medium (MS, 1/2 macronutrients) containing 3 mg/L indolebutyric acid (IBA) and 0.1 mg/L naphthalene acetic acid (NAA) for 15 days. In juvenile material, the best rooting frequencies were achieved by culturing the shoots for 24 or 48 h on Gresshoff and Doy medium (GD, 1/3 macronutrients) containing 25 mg/L IBA. Despite the juvenile character of the donor plants, the rooting rates were below 50% in all genotypes except for in one genotype, in which 96% rooting was observed. Somatic embryogenesis was initiated in shoot tip and leaf explants excised from axillary shoot cultures of both juvenile and mature material previously established in vitro. The SE induction medium consisted of MS supplemented with high levels of auxin (4 mg/L of indol acetic acid or NAA) or even in a medium devoid of plant growth regulators (PGRs). A strong interaction between auxin and genotype was observed for somatic embryo induction. Furthermore, the induction rate was also significantly affected by the type of initial explant chosen, with shoot apices being the most reactive explants in all genotypes. Embryogenic competence was maintained by secondary embryogenesis regardless of the origin (mature or juvenile) of somatic embryos, following the culture of nodular embryogenic structures on Schenk and Hildebrandt medium without PGRs. For plant regeneration, cotyledonary-stage embryos were isolated from embryogenic cultures and stored in empty Petri dishes at 4°C for two months before transfer to germination medium consisting of GD medium supplemented with 0.1 mg/L BA and 20 μM STS. Plant conversion frequencies were 21–67% depending on the genotype, regardless of the juvenile or adult origin. Finally, medium-term conservation of the holm oak germplasm was achieved by applying slow growth procedures to axillary shoot cultures. To reduce growth, cold storage at 4°C under dim lighting was used, which allowed successful conservation of the shoots for 12 months.
Databáze: OpenAIRE
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