The expression analysis of AtRKD4 transgene in Dendrobium lineale Rolfe transgenic orchid carrying 35S::GR::AtRKD4 for micropropagation

摘要

Dendrobium lineale Rolfe is an endemic orchid of Papua, Indonesia. Mass propagation of D. lineale has been performed through the induction of somatic embryogenesis by inserting AtRKD4 embryo gene from Arabidopsis thaliana. This research objective is to confirm whether the AtRKD4 in T-DNA that harbor 35S::GR::AtRKD4 can express properly in D. lineale transnsformant in order to induce somatic embryos (SEs). The leaves from 10 plantlets were cultivated in New Phalaenopsis (NP) medium added with 15 μM Dexamethasone (DEX) or 3 mg.L~(-1) Thidiazuron (TDZ) for SE induction and NP0 as control. The expression was analyzed in developmental stage of 1-, 3-, 5-days and 7-days after induction by DEX and TDZ. The expression of AtRKD4 in orchid genome was detected by Polymerase Chain Reaction (PCR) using AtRKD4 specific primers. The result showed that the transgene expression was detected as 382 bp fragment of cDNAs amplified from transformants after 1-, 3-, 5-and 7-days induction by DEX and TDZ, with the highest expression was after 5 days induction, and probably will induce somatic embryogenesis from leaves. In conclusion, AtRKD4 transgene can be activated by using DEX and TDZ after 1-7-days cultivation with the optimal induction in 5 days.