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#plant transformation

5 public questions tagged with this topic.

Ti plasmid vector commonly used for antisense constructs is:

Transformation of tomato for delayed ripening relies on Agrobacterium tumefaciens binary vector system exemplified by pBIN19. Native Ti plasmid C58 type is ~200 kb containing T-DNA bordered by 25 bp imperfect direct repeats and vir regulon encoding VirA sensor kinase, VirG response regulator, VirD1/D2 endonuclease, VirB T4SS pilus and VirE2 single-stranded DNA binding protein. To facilitate cloning, large plasmid split into helper Ti plasmid disarmed lacking oncogenes iaaM, iaaH, ipt responsible for auxin cytokinin overproduction causing crown gall, retaining vir genes, and small shuttle vector pBIN19 carrying replication origins ColE1 for E. coli and RK2 for Agrobacterium, kanamycin resistance nptIII for bacterial selection, left and right borders, multiple cloning site with lacZ alpha for blue-white screening adjacent to CaMV35S promoter and nos terminator, plus plant selectable nptII driven by nos promoter and marker. Antisense PG cDNA cloned in reverse orientation between promoter and terminator mobilized to Agrobacterium via triparental mating, acetosyringone induced vir expression nicked borders, T-strand transferred integrated via nonhomologous end joining semi-randomly. pBIN19 advantage over pUC19, pBR322 lacking T-DNA and plant signals.

Ref: Bevan M 1984 Nucleic Acids Res 12:8711 pBIN19; Gelvin SB Microbiol Mol Biol Rev Agrobacterium Ti.

Which is NOT required for Agrobacterium-mediated plant transformation in the lab?

Laboratory plant transformation uses disarmed Ti plasmids retaining only essential transfer functions. Vir genes of Ti plasmid sense acetosyringone and produce nickase, chaperones, and secretion apparatus. Type IV secretion system encoded by virB operon plus coupling factor VirD4 forms trans-kingdom channel for nucleoprotein export. Host plant genes encoding attachment receptors, karyopherins, histones, and DNA repair enzymes mediate nuclear targeting and integration. Opine catabolism genes encode transporters and dehydrogenases allowing bacteria to utilize opines as nitrogen and carbon source in crown gall niche. This nutritional advantage is irrelevant on synthetic culture media, making it dispensable.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.