Castor (Ricinus communis L.) is a high-yielding oilseed crop native to tropical Africa. The seed contains ~60% oil by weight, yielding approximately 1,200 kg of oil per hectare. The oil is composed of ~90% ricinoleic acid, a unique hydroxylatty acid. Its unique composition provides castor oil with distinctive characteristics important for industrial use. Unfortunately, this valuable oilseed has not been widely cultivated in the United States since 1972, due in part to the presence of ricin in the seed. Ricin is a highly toxic lectin found in the endosperm of mature castor seed. This project sought to silence ricin production through the introduction of an RNAi element into the castor genome. The RNAi vector (pC1-RKO) containing a segment of ricin mRNA and its inverted repeat separated by a chalcone synthase A intron from pFGC5941 enclosed in a pCambia1301 backbone was created, verified via sequencing, and transformed into Agrobacterium tumefaciens for castor transformation. Fungal contamination was a serious concern; successful disinfestation used a 10-minute wash with 0.1% mercuric chloride (w/v). Media supplemented with 6-benzylaminopurine generated healthier shoots from embryo axes dissected from mature seed compared to thidiazuron-treated mesocotyls dissected from mature seed. Short treatments of thidiazuron on 6-benzylaminopurine initiated shoot cultures showed greater shoot proliferation on embryo axes dissected from mature seed. Rooting occurred with incubation on half-strength medium containing naphthaleneacetic acid or indole-3-butyric acid; however, naphthaleneacetic acid produced hardier roots which better survived acclimatization. Inoculation of embryo axis explants after 2 days pre-culture improved survivability. Likewise, transformations using A. tumefaciens cultures of 0.5 O.D.600 and lower did not lead to downstream bacterial contamination. The pCambia1304 vector was used as a test plasmid for refinement of the transformation protocol. Of the 870 pCambia1304 inoculation explants, 2 survived hygromycin screening and showed gusA activity. Of the 2,500 pC1-RKO inoculated explants, 6 survived hygromycin selection and rooted. Further analysis via PCR, end-point RT-PCR, and Western and dot-blotting showed these to be non-transformed and ricin content unaffected.
| Identifer | oai:union.ndltd.org:MSSTATE/oai:scholarsjunction.msstate.edu:td-1477 |
| Date | 13 December 2014 |
| Creators | Barnes, Daniel Joseph |
| Publisher | Scholars Junction |
| Source Sets | Mississippi State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Theses and Dissertations |
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