Molecular characterization of transgenic sweet potatoes (ipomoea batatas(L). Lam.) Following genetic transformation with viral coat proteins and gus genes
| dc.contributor.advisor | Machuka, Jesse | |
| dc.contributor.advisor | Nkanata, Gitonga | |
| dc.contributor.advisor | Gichuki, Simon | |
| dc.contributor.author | Mutegi, Rosemary Wanja | |
| dc.date.available | 2012-01-03T13:31:38Z | |
| dc.date.issued | 2012-01-03 | |
| dc.description | Department of Biochemistry and Biotechnology, 89p. The SB 211.S9M8 2005 | |
| dc.description.abstract | Sweet potato (ipomoea batatas (L.) Lam) is a major crop that ranks seventh among food crops in annual production in the world. The most widespread virus infecting sweet potato is the sweet potato feathery mottle virus (SPFMV). Yield loss caused by the virus can be as high as 80%. Efficient methods to control the virus are not available and conventional breeding programs to introgress genetic resistance into the cultivated germplasm have had very limited success. Breeding for resistance through genetic engineering offers an alternative solution for the control of SPFMV. Collaborative efforts between Monsanto Company in the USA and the Kenya Agricultural Research Institute (KARI) succeeded in introducing SPFMV coat protein gene in an attempt to enhance resistance to SPEMV in the sweet potato. Molecular analysis of transgenenics as well as expression studies haven't been carried out. In order to do this, the following work was carried out to confirm the presence, expression and the number of copies of the transgene inserted using an Agrobacterium tumefaciens medicated system. Kanamycin painting showed that leaves of transgenic plants failed to show visible damage. Those of the non-transformed control plants treated with kanamycin exhibited severe necrosis, chlorosis and bleaching. There were no differences in antibiotic resistance at 1.5% kanamycin concentration between the transgenic lines. These transgenic sweet potato plants grew normally and formed storage roots after three months in the screen house. There were no morphological differences between the untransformed control plants and the transgenic plants when they were grown in the screen house. Leaves of the freshly sprouting shoots from harvested storage roots of transgenic plants also showed kanamycin resistance suggesting that the antibiotic resistance gene was transmitted to their vegetatively propagated progenies through storage roots. Up to eighty five percent of the transgenic sweet potato explants analyzed histochemically had a characteristic blue staining of the entire surface. Wounded (cut) regions on the leaf reacted moreintensively, suggesting higher enzymatic activity. The roots displayed intense blue colour in the whole segment tested indicating strong gene activity. Amplified DNA fragments of 450bp were produced in 65% of the samples tested using coat protein specific primers and DNA extracted from putative transgenic plants. This 450bp fragments was the size of the coat protein gene that had been inserted in the sweet potato using Agrobacterium as a vector. No amplification occured with the DNA from control plants and in two putative transformants. The transgene in these two putative transformants might have been silenced or undergone some positional effects. Southern blot analysis revealed that the plasmid DNA had a single copy insert. This positive control sample produced a light diffuse band indicating a single copy insert. There were no bands detected in the other transformants. This was attributed to the insensitive detection method used. From this study, simple efficientand cost effective methods of confirming transformability in transgenics have been developed which are being applied in other transgenic crops. Some sweet potato lines characterized in this study were also shown to have integrated the transgene stably in their genome, which is likely to reduce yield losses if the gene will be translated to functional proteins by the plant cell ribosomes.A | en_US |
| dc.description.sponsorship | Kenyatta University | en_US |
| dc.identifier.uri | http://ir-library.ku.ac.ke/handle/123456789/2220 | |
| dc.language.iso | en | en_US |
| dc.subject | Sweet potatoes--Genetics | |
| dc.subject | Transgenic plants | |
| dc.subject | Ipomoea | |
| dc.title | Molecular characterization of transgenic sweet potatoes (ipomoea batatas(L). Lam.) Following genetic transformation with viral coat proteins and gus genes | en_US |
| dc.type | Thesis | en_US |