The correct answer is (3) A, C and D.

Transgenic and non‑transgenic plants of the same variety (and with unknown insertion site) are best distinguished by:

• a visible/selectable marker phenotype (A),

• Southern hybridization with a probe to the transgene (C), and

• PCR using transgene‑specific primers (D).

PCR with host‑genome primers alone (B) cannot distinguish them because host DNA is identical in both.

Option-wise explanation

A. Phenotype linked to the marker gene – Useful

• If the construct carries a selectable or screenable marker (e.g., antibiotic/herbicide resistance, GFP), only transgenic plants will show the phenotype.

• This provides a straightforward first‑level distinction between transgenic and non‑transgenic individuals.

B. PCR using primers specific to the host genome – Not useful alone

• Host‑genome primers amplify the same locus in both transgenic and non‑transgenic plants of the same variety.

• Unless the insertion site within that locus is already known (it is stated to be unknown), these primers will not differentiate the two groups.

C. Southern hybridization – Useful

• Genomic DNA is digested and probed with a transgene‑specific probe.

• Bands appear only in plants carrying the transgene; the number and size of bands reflect insertion presence and copy number.

• Site of insertion need not be known beforehand.

D. PCR using transgene-specific primers – Useful

• Primers designed to the transgene sequence amplify a product only if that sequence is present in the genome.

• Transgenic plants yield a band; non‑transgenic plants do not, regardless of where the T‑DNA inserted.

Why combination A, C and D is correct

• A gives phenotype-based evidence of transformation.

• C and D provide molecular confirmation independent of insertion site.

• B adds nothing when insertion site is unknown.

Therefore, the most appropriate combination to distinguish transgenic from non‑transgenic plants is (3) A, C and D.