The correct combination is (4) B and D. Disarmed Ti plasmids are used and the oncogenes (tumor‑inducing genes) have been removed, so no crown‑gall tumors form in transgenic plants.

Concept: disarmed Ti plasmid

In nature, the T‑DNA of the Ti plasmid carries oncogenes for auxin and cytokinin biosynthesis plus opine synthase genes, which cause uncontrolled cell division (tumor) and opine production. In plant genetic engineering, this T‑DNA is disarmed: the oncogenes are deleted and replaced with the desired transgene plus selectable markers, while the vir region on a helper plasmid still mediates T‑DNA transfer. As a result, plant cells receive foreign DNA but no tumor‑inducing functions, so they regenerate into normal, transgenic plants.

Statement-by-statement analysis

A. Vir D2 gene is mutated in Ti plasmid.

• VirD2 is a virulence protein required for nicking the T‑DNA borders and covalently binding to the 5′ end of the T‑strand; it is essential for efficient T‑DNA transfer and integration.

• In standard transformation systems VirD2 remains functional; mutating it would drastically reduce transformation efficiency, not just tumor formation. This is not the usual reason tumors are absent, so A is incorrect.

B. Disarmed Ti plasmid is generally used.

• True. “Disarmed” means the T‑DNA region no longer carries oncogenes but retains border sequences where the transgene cassette is inserted.

• Using such a plasmid allows T‑DNA transfer without tumorigenesis, directly explaining why no crown‑gall disease appears in transgenic plants. B is correct.

C. Heat-shock during transformation destroys virulence.

• Brief heat treatments used in some protocols are typically for bacterial or plant handling (e.g., competence induction in other systems), but Agrobacterium transformation itself does not rely on heat‑shocking bacteria to abolish virulence.

• Virulence is controlled genetically (by deleting oncogenes), not by transient heat shock, so C is incorrect.

D. Oncogenes have been removed.

• True and central to the logic: deleting auxin–cytokinin oncogenes from T‑DNA prevents tumor formation even though T‑DNA still integrates.

• This is exactly how Ti plasmids are converted into safe cloning vectors, so D is correct.

Option evaluation

• (1) A and C – Both incorrect explanations.

• (2) A and D – A is wrong; only D is right.

• (3) B and C – C is wrong; eliminates this choice.

• (4) B and D – Both match how modern binary/disarmed Ti systems work, so this is the correct answer.

SEO‑oriented introduction (for article use)

Agrobacterium tumefaciens is called a natural genetic engineer because it can transfer T‑DNA from its Ti plasmid into plant genomes, causing crown‑gall tumors in the wild. In plant biotechnology, however, researchers use disarmed Ti plasmids in which the tumor‑inducing oncogenes have been removed and replaced with useful transgenes, so plants become transgenic without developing galls, making statements B and D the correct combination.