Production of Doubled Haploid Embryos from Cork Oak Anther Cultures by Antimitotic Agents and Temperature Stress

Abstract

Cork oak (Quercus suber L.) is a forest tree species of the family Fagaceae. It is characterized by long life cycles which hamper doubled haploid plant production to obtain homozygotes and pure lines. The time-consuming method of repeated backcrossings by conventional breeding techniques to produce pure lines is impractical in woody species. Nevertheless, biotechnology has offered new tools to make it possible. A doubled haploid plant or embryo is one that is developed by the doubling of a haploid set of chromosomes. A protocol to produce doubled haploids of cork oak has been developed through microspore embryogenesis. By a heat stress treatment, the microspores inside the anther leave the gametophytic pathway and react shifting their development to the sporophytic pathway by means of which haploid embryos are obtained. Chromosome duplication of haploids from cork oak anther cultures occurs either spontaneously or may be induced by the application of antimitotic agents (e.g., colchicine, oryzalin, amiprophos-methyl). Furthermore, a genetic test is designed through microsatellite markers to elucidate whether the diploid embryos obtained are originally haploids which spontaneously duplicated their genome, or alternatively those embryos are generated from the diploid tissue of the anther wall. Here we describe a detailed protocol to produce doubled haploid individuals from cork oak anther cultures by using temperature stress and antimitotic agents.

Keywords: Amiprophos methyl; Colchicine; Flow cytometry; Microsatellite markers; Microspore embryogenesis; Oryzalin; Quercus suber; Spontaneous diploidization.