iv
ABSTRACT
GENETIC TRANSFORMATION OF LENTIL ( Lens culinaris M. cv.Sultan.1)
WITH A TRANSCRIPTION FACTOR REGULATOR (MBF1c)
AND
ANALYSIS OF TRANSGENIC PLANTS
KAMÇ / I, Hamdi
Ph.D., Biotechnology, Institute of Natural ad Applied Sciences
Supervisor Prof. Dr. Meral YÜ / CEL
Co-Supervisor: Dr. Ufuk Ç / elikkol AKÇ / AY
September 2011, 252 pages
In this study, Agrobacterium mediated genetic transformation of lentil Sultan 1
cultivar with MBF1c and evaluation of transgenic plants was aimed.
The study was initially based on optimized protocol with Agrobacterium tumefaciens
KYRT1 strain and pTJK136 binary plasmid. Based on this protocol and transient
marker gene expression in embryo apex, 15% stable transformation efficiency was
aimed. However limited knowledge about pTJK136 and problem with curing KYRT1
leaded us to use Agrobacterium tumefaciens C58C1 strain and also to engineer an
alternative binary plasmid / pPZP101. Hence, scope of this study became construction
of a plant binary transformation vector and lentil transformation optimization with
C58C1 strain.First plant transformation vector designed in this study was pPZP101ManA-MBF1c.
Transformations with C58C1::pPZP101ManA-MBF1c were carried out with a
reformulated co-cultivation media. Cotyledonary nodes were isolated from three
days old lentil seedlings germinated with phytormone (BAP/TDZ) induction. Isolated
nodes were either injured and pre-incubated in co-cultivation media or pre-
incubated and then injured prior to transformation. Regeneration and necrosis
behaviors of the transformed explants leaded us to the conclusion that explant
preparation is the critical step of transformation. And data suggest that explants
isolated from 2mg/l BAP, pre-incubated two days in co-cultivation media, injured
and transformed performed significantly better scores for necrosis shoot
regeneration and callus formation parameters.
Transformed explants that survived in subsequent sub-cultures in mannose selection
raised shoots. These shoots were grafted and regenerated into plantlets. The
putative transgenic plantlets were screened for transgene with PCR. Initial
amplification signals fainted and lost as grafts grew. In order to make a diagnosis of
this fainting behavior the second plant transformation vector pPZP101ManA-
GUSint-MBF1c was constructed and transient GUS expression analysis were made.
| Identifer | oai:union.ndltd.org:METU/oai:etd.lib.metu.edu.tr:http://etd.lib.metu.edu.tr/upload/12613845/index.pdf |
| Date | 01 September 2011 |
| Creators | Kamci, Hamdi |
| Contributors | Yucel, Meral |
| Publisher | METU |
| Source Sets | Middle East Technical Univ. |
| Language | English |
| Detected Language | English |
| Type | Ph.D. Thesis |
| Format | text/pdf |
| Rights | To liberate the content for public access |
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