The correct combination is (4) A, C and D. Statements A, C, and D are true, while B is false.​

Explanation of each statement

A. A. tumefaciens is a gram-negative soil bacterium – TRUE

• Agrobacterium tumefaciens is a rod‑shaped, Gram‑negative bacterium commonly found in the rhizosphere of plants and in agricultural soils.​

B. Opine catabolism genes are present in T‑DNA region of Ti‑plasmid – FALSE

• The T‑DNA region contains genes for auxin, cytokinin and opine biosynthesis (e.g., ocs, nos), which are transferred into the plant genome.

• Opine catabolism (uptake and degradation) genes are located in distinct catabolic regions (occ, noc, etc.) on the Ti plasmid but are outside the T‑DNA, remaining in the bacterium.​

C. Opines are synthesized by condensation of amino acids and α‑ketoacids or amino acids and sugars – TRUE

• Chemically, most opines are formed by reductive condensation of an amino acid with either an α‑keto acid or a sugar/carbonyl compound, giving nopaline/octopine‑type or mannityl/chrysopine‑type opines.​

D. A callus culture of crown gall tissue caused by A. tumefaciens in plants can be multiplied without adding phytohormones – TRUE

• Crown gall cells harbor integrated T‑DNA that encodes auxin and cytokinin biosynthesis, so they synthesize hormones autonomously and grow in vitro without exogenous phytohormones, a classic hallmark of transformed gall tissue.​

Option-wise evaluation

• (1) A, B and C – Incorrect because B is false.

• (2) A, B and D – Incorrect because B is false.

• (3) B, C and D – Incorrect because B is false and A (true) is omitted.

• (4) A, C and D – Correct; all three statements accurately describe A. tumefaciens, opine chemistry and hormone‑independent crown gall callus growth.​

SEO‑oriented introduction (for article use)

Agrobacterium tumefaciens is a Gram‑negative soil bacterium whose Ti plasmid T‑DNA causes hormone‑autonomous crown gall callus and opine synthesis in infected plants, while separate opine catabolism genes on the plasmid allow the bacterium to use opines as nutrients. In exam questions, the correct combination of facts is that A. tumefaciens is Gram‑negative, opines arise from condensation of amino acids with α‑ketoacids or sugars, and crown gall callus can be subcultured without added phytohormones, giving option (4) A, C and D as the right answer.