Memorias de investigación
Book chapters:
Protocol for doubled-haploid micropropagation in Quercus suber L. and assisted verification.

Research Areas
  • Agriculture

Forest trees are characterized by long life cycles which hamper doubled-haploid plant production to obtain homozygotes and pure lines. Nevertheless, Biotechnology has offered new tools to make it possible. We present a new protocol to obtain doubled-haploids (DH) of cork oak (Quercus suber L.), from induction to plantlet regeneration. A correlation between the phenologic traits of catkins, the size and colour of anthers and the developmental stage of the microspores has been established. Gametic development was diverted to the sporophytic pathway by stress treatments, and a chilling pre-treatment of the catkins. Genotype also affected the induction of pollen embryogenesis. Cellular characterization of the first stages of microspore embryogenesis and embryo development have been studied. Ploidy level of the cork oak embryos induced from anther cultures was verified by flow cytometry. The haploid genotype was the most frequently observed, although a small percentage of diploid embryos was also found. Microsatellite markers permitted the verification of the gametic origin of those anther embryos which had diploid genome. Spontaneous diploidization of anther embryos is a rare phenomenon in forest species. As a consequence, the regeneration of DH plants has to be obtained through treatments with antimitotic agents, such as colchicine, oryzalin and amiprophos-methyl. Both viability and ploidy level of the embryos treated with antimitotic agents were tested. Oryzalin and amiprophos-methyl 10 µM were more efficient than colchicine in genome diploidization. Maturation in medium supplemented with 1% activated charcoal provided a significant increase in embryo size and weight. Stratification at 4 ºC for two months in the same maturation medium favoured the germination on medium supplemented with 6-benzyl-adenine and indole-3-butyric acid, and the subsequent acclimation to soil. The aim of the present investigation is to develop an efficient protocol to obtain double-haploid plants of cork oak. At present, those plants have been established in a field trial and are subjected to a progeny test.
Book Edition
Book Publishing
Book title
Protocols for Micropropagation of Woody Trees and Fruits
From page
To page

Research Group, Departaments and Institutes related
  • Creador: Grupo de Investigación: Tecnologías y Métodos para la Gestión Sostenible
  • Departamento: Silvopascicultura