454 GS FLX+ sequencing; Comparative 454 pyrosequencing of transcripts to provide a resource for the study of embryogenesis of coconut (Cocos nucifera L.)
Comparative 454 pyrosequencing of transcripts to provide a resource for the study of embryogenesis of coconut (Cocos nucifera L.)
Microspore derived embryos (MDE) were collected from in vitro grown anthers. MDE were induced on Eeuwens Y3 (Karunaratne et al. 1985) medium supplemented with 100 micromolar 2, 4-dichlorophenoxyacetic acid and 100 micromolar 1-naphthalene acetic acid (Perera et al. 2008). Total RNA was extracted using an RNeasy Plant Mini Kit (Qiagen, UK) using manufacturers protocol. cDNA libraries were constructed using SMART approach as described by Zhu et al. 2001 using the the oligonucleotides SMART Sfi1A : 5prime- AAG CAG TGG TAT CAA CGC AGA GTG GCC ATT ACG GCCrGrGrG-3', CDS-Sfi1B : AAG CAG TGG TAT CAA CGC AGA GTG GCC GAG GCG GCCd(T)20-3',  SMART PCR primer : 5'-AAG CAG TGG TAT CAA CGC AGA GT- 3'. The amplified cDNA PCR products were purified using QIAquick PCR Purification Kit (QIAGEN, CA) and a final  yield of 3 microgram was prepared for each tissue type.