National Academy of Agricultural Sciences (NAAS)
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PRINT ISSN : 2319-7692
Online ISSN : 2319-7706 Issues : 12 per year Publisher : Excellent Publishers Email : editorijcmas@gmail.com submit@ijcmas.com Editor-in-chief: Dr.M.Prakash Index Copernicus ICV 2018: 95.39 NAAS RATING 2020: 5.38 |
Transferring sesquiterpenoid biosynthetic genes into a high-biomass crop has been proposed as one route to broadening the production base for plant-derived pharmaceutical compounds, which are frequently obtained from low-yielding source species. Cotton is an attractive candidate host because of its biomass, its non-food end use and its established tissue-culture systems, yet it has received little attention in this context and is known to be recalcitrant in culture. This study reports the design of a binary overexpression construct carrying a sesquiterpene synthase coding sequence and its stable introduction into cotton. The 1,641 bp coding sequence was placed between a constitutive cauliflower mosaic virus 35S promoter and a Rubisco small-subunit terminator using XbaI and NcoI sites, and the cassette was assembled into the pBINPLUS backbone together with an nptII selectable marker, giving a construct of 15,709 bp. The construct was introduced into Escherichia coli DH5α and then into Agrobacterium tumefaciens LBA4404; colony PCR and restriction analysis confirmed its integrity at each step. Hypocotyl explants of cv. Coker-312 were co-cultivated with the recombinant bacterium, and transformants were recovered on kanamycin-containing media through callus induction, somatic embryogenesis and plantlet conversion. Control explants inoculated with sterile water did not survive selection. The complete cycle from explant to acclimatized plant occupied approximately eight to nine months, and two independent lines were confirmed by PCR and Sanger sequencing, with BLAST analysis indicating complete identity between the integrated and donor sequences. Artemisinin and its precursor arteannuin B were subsequently detected in leaf material of these lines by UHPLC-QTOF-MS, as we reported previously in two national-language publications; the present article provides the full English-language account of the construct design and of the transformation and regeneration protocol, with the expanded methodological detail required for the procedure to be reproduced.
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