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Protocols for Micropropagation of Woody Trees and Fruits

Protocols for Micropropagation of Woody Trees and Fruits

Protocols for Micropropagation of Woody Trees and Fruits

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DOUBLED-HAPLOID MICROPROPAGATION IN QUERCUS SUBER L.<br />

Figure 6. Scheme <strong>for</strong> the germination <strong>of</strong> cork oak doubled-haploid embryos. A) Mature<br />

doubled-haploid embryos. B) Germinated doubled-haploid embryos.<br />

2.8.1. Verification through Microsatellite Markers<br />

Extraction <strong>of</strong> leaf DNA. First, DNA is extracted from leaves <strong>of</strong> the parent trees,<br />

following the protocol described by Ziegenhagen et al. (1993). DNA samples were<br />

taken from 12 trees.<br />

1. About 200 mg <strong>of</strong> leaf simples are placed in a sterile Eppendorf tube <strong>and</strong> 1<br />

ml extraction buffer is added (100 mM sodium acetate pH 4.8, 50 mM<br />

EDTA pH 8, 500 mM NaCl <strong>and</strong> 2% polyvinyl pyrrolidone, PVP). The buffer<br />

pH is adjusted at 5.5 <strong>and</strong> 1.4% sodium dodecyl sulfate (SDS) is added. The<br />

mixture is grinded with a sterile stick.<br />

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