Holm Oak Somatic Embryogenesis: Current Status and Future Perspectives

Abstract

Quercus ilex (holm oak) is one of the most representative trees in the Mediterranean basin, but now the sustainability of its ecosystems is at serious risk due to the lack of natural regeneration and to the presence of a severe disease called oak decline that has caused the death of thousands of trees. The application of biotechnological tools, such as somatic embryogenesis, allows programs of genetic improvement of the species to be speeded up and helps in the conservation of its ecosystems. Somatic embryogenesis is currently considered one of the main biotechnological techniques that has demonstrated significant benefits when has applied to forest tree species, providing significant advantages such as mass propagation, genetic transformation, application of synthetic seed, and cryopreservation of elite genotypes. In this report, the state of the art of somatic embryogenesis in holm oak is reviewed. Factors affecting the induction (plant donor age, type of explant, or plant growth regulators) and maintenance and proliferation of the embryogenic cultures are summarized. Advances on the conversion of somatic embryos into plants and on the acclimatization of these plantlets, as well as the results obtained on the application of the genetic transformation and the cryopreservation procedures to holm oak embryogenic cultures, are also presented.

Keywords: Quercus ilex; cryopreservation; genetic transformation; oak decline; plant regeneration; somatic embryos.

Figure 1

Somatic embryogenesis induction on different explants derived from adult trees of Quercus spp. (A,B) Somatic embryos formed on leaf explants excised from in vitro shoot cultures of Q. robur (A) and Q. alba (B). (C) Globular-stage somatic embryo generated on shoot tip of Q. rubra. (D,E) Somatic embryos induced on apex (D) and leaf (E) explants excised from axillary shoot cultures of Q. ilex. (F) Embryogenic callus generated from a male catkin of Q. ilex. Scale bar: 1 mm.

Figure 2

Somatic embryo proliferation and plant regeneration on holm oak. (A) Morphological aspect of nodular embryogenic structures used as inocula explants in proliferation, cryopreservation, and genetic transformation experiments. Scale bar: 1 mm. (B) Somatic embryos originated from nodular embryogenic structures showed in A after 6 weeks of culture on proliferation medium. (C) Morphological aspect of 2-month chilled mature somatic embryos. (D,E) Plantlets regenerated from somatic embryos subjected to cold treatment for 2 months and 6 weeks in germination medium. (F) Plantlet following two seasons of growth in the greenhouse.

Figure 3

Cryopreservation and genetic transformation of embryogenic cultures of holm oak. (A) Somatic embryo clumps generated from cryopreserved nodular embryogenic structures following exposure to PVS2 solution for 15 min and 6 months in LN. (B) High magnification of A (square) to show somatic embryos generated from a cryopreserved nodular structure. Scale bar: 1 mm. (C) Transgenic somatic embryos and nodular embryogenic structures after transformation with EHA105pTAU strain and 10 weeks on kanamycin medium. Scale bar: 1 mm. (D) Somatic embryos showing green fluorescence under UV light.