Tissue culture, ultrastructural studies and essential oil analysis of Ocimum basilicum L. / Aziemah Abdul Manan

Aziemah, Abdul Manan (2017) Tissue culture, ultrastructural studies and essential oil analysis of Ocimum basilicum L. / Aziemah Abdul Manan. Masters thesis, University of Malaya.

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Abstract

This study describes efficient protocols for coloured callus formation, in vitro shoot multiplication, complete plant regeneration, and in vitro flowering from leaf and shoot tip explants of Ocimum basilicum ‘Sweet Thai’, chemotype methyl chavicol. In addition, it reveals information on pollen and seed germinability, non-glandular and glandular trichome ultrastructures, and essential oil content and composition of the plant at different plant developmental stages. Leaf and shoot tip explants from two-month-old aseptic seedlings were induced to form coloured callus, multiple shoots, complete plants, and in vitro flowers on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) [6-benzyl-aminopurine (BAP) and gibberellic acid (GA3) either alone or in combination with α-naphthaleneacetic acid (NAA) or 2,4- dichlorophenoxyacetic acid (2,4-D)]. The highest percentage of purple callus formation (100%) from both leaf and shoot tip explants was recorded on MS medium supplemented with 0.5 mg l-1 BAP and 0.5 mg l-1 NAA and also on MS medium supplemented with 1.0 mg l-1 BAP and 0.5 mg l-1 NAA. Whilst, the highest percentage of light green callus formation (100%) from both leaf and shoot tip explants was recorded on MS medium supplemented with 0.5 mg l-1 BAP and 1.0 mg l-1 2,4-D and also on MS medium supplemented with 1.0 mg l-1 BAP and 1.0 mg l-1 2,4-D. The highest number of multiple shoots (mean 5 shoots per explant) was obtained from shoot tip explants cultured on MS medium supplemented with 1.0 mg l-1 BAP. Multiple shoots showed 100% rooting on half-strength MS basal medium. Shoot tip explants exhibited the highest percentage of in vitro flowering (40%) on MS medium supplemented with 1.0 mg l-1 GA3. All (100%) of the multiple shoots flowered in vitro on MS medium supplemented with 1.0 mg l-1 GA3. The rooted plantlets were successfully acclimatized in vermiculite and established in a soil mixture (3 garden soil: 2 potting mix) in a field with an 80% survival rate, which further developed flowers ex vitro after 24 weeks. Relative to the mother plant, in vitro grown plants flowered at a younger stage of plant development but produced no seeds, and showed a lack of pollen germination, fewer fully-filled peltate glandular trichomes, lower essential oil content, and higher methyl chavicol content. Ex vitro grown plants flowered at an intermediate stage of plant development with seed formation, and showed nearly the same pollen and seed germinability, essential oil content, and methyl chavicol content as in the mother plant.

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