Abstract:
Orchidaceae is one of the largest and most diverse families of flowering plants and a
major export ornamental crop. Cultivar development is the key for the success of
ornamental flower industry and therefore, it is vital to identify the genetic diversity
of the cultivars. In such an attempt, the first step is to optimize basic molecular
biological techniques involved in genetic diversity analysis. However, DNA
extraction from orchids is challenging compared to the other plant species since
orchids are rich in polysaccharides and secondary metabolites, which can act as
inhibitors in downstream applications. Most of the standardized protocols require
liquid nitrogen freezing step, which is not an affordable practice in the laboratories
of developing countries. Therefore, optimization of a low cost protocol to obtain pure
DNA is necessary. The objective of the current study was to optimize extraction of
genomic DNA for molecular marker based genetic diversity studies of selected
orchid cultivars with ornamental value. Leaf pieces and pestle and mortar were stored
at -80°C for at least three days. DNA extraction was done from frozen orchid leaves
(50-100 mg young leaves) using Promega Wizard genomic DNA purification kit
(Promega Inc. USA), classical CTAB protocol and modified CTAB protocol. Except
modified CTAB protocol, none of the other methods produced high quality DNA as
determined by spectrophotometry and agarose gel electrophoresis. However, the
method was successful only for the Dendrobium cultivars but not for the
Phalaenopsis leaves tested. Successful amplification of orchid rDNA-ITS region
confirmed that the quality of extracted DNA is suitable for other PCR based
molecular marker studies such as Random Amplified Polymorphic DNA (RAPD)
and Simple Sequence Repeats (SSRs). The modified method was reliable and
reproducible.
