Abstract:
Bitter gourd is a monoecious crop that belongs to the Curcubitaceae family. It has
significant agricultural and medicinal value. High levels of bioactive substances such
as phenolic compounds and polysaccharides, along with other contaminants, restrict
obtaining high-quality and higher-quantity DNA from these plants. To minimize
contaminants and to ensure the integrity of extracted DNA, extraction protocols must be
modified. The aim of this study is to optimize the DNA extraction protocol for Mormodica
charantia by using variations of the CTAB method. This protocol mainly depends on
the buffer that is used to lyse cells and separate DNA from other cellular compounds
with phenol, chloroform, and isoamyl alcohol. Three variations of the protocol were
followed. Tender leaves were used for DNA extraction. UV spectrophotometry was used
in the following wavelengths, A230, A260, A280, and A320, to evaluate the quantity
and quality of the isolated DNA obtained through each protocol, and the results were
compared. The results from UV spectrophotometry were further validated by agarose
gel electrophoresis. The first protocol was the standard CTAB extraction method,
which yielded 0.884±0.245 (μg/150mg) DNA with a concentration of 17.68±4.89 (ng/
μl). The second protocol was the modified CTAB with cold treatment. Leaves were
ground in a cold motor and pestle, which was surrounded by ice cubes, that yielded
0.3±0.079 (μg/150mg) DNA with a concentration of 5.994±1.582 (ng/μl). The third
protocol was modified CTAB with cold treatment and RNAse treatment; this method
yielded 0.461±0.265 (μg/150mg) DNA with a concentration of 13.23±6.92 (ng/μl). The
optimized protocol 3 was found to be more suitable and comparatively gave high yields
of pure DNA with minimal RNA contamination in the presence of the RNAse enzyme,
as ensured by Agarose gel electrophoresis. Furthermore, the DNA templates generated
using the modified method could facilitate genetic studies, marker-assisted breeding,
and molecular breeding efforts in bitter gourd to enhance desirable traits in the future.
This protocol can be further refined and applied to other plants that have similar
biochemical properties.