Establishment Of In Vitro Plantlets Of Artemisia Annua L. For The Analysis Of Artemisinin Biosynthetic Gene (CYP71AV1) And Trichome Initiation Gene (GL3)
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Date
2014-06
Authors
Suganthi Appalasamy
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Abstract
Artemisia annua L. is an herb known for its secondary metabolite, artemisinin. Artemisinin is used as antimalarial drug but its availability is limited by low yield in plantation. To produce artemisinin using in vitro technique, a high yielding in vitro cultivar must first be selected. For the establishment of aseptic seedlings of three selected clones of Artemisia annua L., the seeds were surface sterilized with 10% (v/v) Clorox® for five minutes followed by 70% (v/v) ethanol for five minutes. This sterilization protocol enabled the establishment of 96.7 % aseptic seeds for TC1 and TC2 clones, and 86.7 % for the Highland clone. The percentage of seed germinated for all the clones were found to be in the range of 13.3 to 36.7 %. Imbibitions test on the three clones of A. annua L. seeds indicated imbibitions before seed germination was not required. The best substrate combination for seed germination of all the three clones of A. annua L. was sand: black soil (1:2) combination. Effect of in vitro and greenhouse condition on A. annua L. plantlets growth indicated in vitro growth condition produced taller plantlets than greenhouse conditions. There were no differences in number of glandular and non-glandular trichome on in vitro and greenhouse grown leaves of A. annua L. The assembly and analysis of transcriptome library using next generation sequencing technology produced 10, 647 gene sequences. Of the 10, 647 genes identified through BLAST software, 306 unique genes of A. annua L. were classified to be involved in cellular function, biological
processes and molecular function. Of the 306 genes, there were 14 unique genes that were identified to be involved in metabolite biosynthesis pathways. CYP71AV1 and GL3 genes that were involved in artemisinin biosynthesis were chosen for expression study of control and mutant plantlets. The mutation density due to ethyl methanesulfonate (EMS) treatment using CYP71AV1 promoter gene was 1 in 408 kb of nucleotides compared to sodium azide induced mutation with 1 in 816 kb. The mutation detection rate for EMS-induced was 2.4 whereas for sodium azide-induced was only 1.2 mutations in every 1000 kb of nucleotides. Shoot tips of all the three clones of A. annua L. treated with 1% EMS showed consistently higher expression level for GL3 gene than in control plantlets. The other plantlets treated with sodium azide were not found to have consistently higher expression level than the control plantlets. GL3 gene expression was found to be a suitable marker in indicating artemisinin yield in A. annua L. initiated from treated shoot tips with 1% EMS.
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Artemisia annua L. is an herb known , for its secondary metabolite, artemisinin.