Research internship on tissue culture and various molecular techniques

Date

12-2014

Degree

Bachelor of Science in Agricultural Biotechnology

College

College of Agriculture and Food Science (CAFS)

Adviser/Committee Chair

Evalour T. Aspuria

Abstract

This internship was conducted to familiarize the student with and gain skills in Plant Tissue culture of Basella rubra L and various biochemical and molecular techniques using various plants. The first part of the study was done to establish sterile cultures of Basella rubra L. from nodal sections, induce shoot and callus formation, and localize anthocyanin from regenerated shoots and callus cultures, whereas the second part was to learn extraction, separation and purification of proteins, DNA and plasmid. Likewise, data gathered from both studies were analyzed, accordingly. Callus formation was induced in culture media containing 2 mg/L BA in combination with either (0.5 mg/L or 1.5 mg/L) 2,4-D or (0.2 mg/L or 0.5 mg/L) NAA. White friable callus proliferated at the upper region of cultures treated with 2 mg/L BA with all levels of 2,4-D and 0.2 mg/L NAA, five to eight days after inoculation. On the other hand, compact callus was noted in cultures inoculated in 2 mg/L BA + 0.5 mg/L NAA, 14 days from inoculation. Violet exudation was observed in culture media that induced callus formation. More intense violet pigmentation was noted in nodal cultures treated with 2 mg/L BA + 0.5 mg/L 2,4-D, and 2 mg/L BA + 1.5 mg/L 2,4-D with moderately dark and dark pigment than cultures supplied with NAA (0.2 mg/L and 0.5 mg/L) which showed very faint and faint pigmentation, respectively. The exudation of violet pigment in the culture media can be associated with the accumulation of anthocyanin which was evident in inner region of nodal section of Basella rubra L. treated with BA + 2,4-D as viewed in stereo microscope. Furthermore, shoots formed in 3-4 days after inoculation in all treatments, except for those inoculated in culture media supplemented with both levels of 2-4-D. Hundred percent (100%) shoot formation was obtained in nodal sections inoculated onto MS medium supplied with BA alone. In the second study, a protease from the leaf of Euphorbia hirta L. was isolated and identified to have an estimate size of >30Kb using SDS-PAGE. Bradford assay determined the protein concentration of 6.66 ug/uL in the leaves of purple-flowered while 7.30 ug/uL in whiteflowered sample. Using the bead-beater in combination with CTAB method, DNA was extracted from 'Lakatan' Banana and Abaca BC2 Hybrid. Plasmids from bacterial colonies which have a molecular weight of approximately 3Kb were also isolated using alkaline lysis method. The DNA insert from the isolated plasmid was determined with molecular weight of 500bp through restriction enzyme digestion.

Language

English

Location

UPLB Main Library Special Collections Section (USCS)

Call Number

LG 993 2014 A127 /C38

Notes

Major in Crop Biotechnology

Document Type

Thesis

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