Biotechnology and its Applications — Class 12 Biology practice questions

Question 1

Read the following passage and answer the questions that follow:

A biotechnology company is developing a transgenic crop to address two simultaneous agricultural problems. The target plant is a soybean variety that suffers heavy yield losses due to (i) attack by a specific lepidopteran pest (pod borer), and (ii) infection by a soil-dwelling parasitic nematode (Heterodera glycines — soybean cyst nematode). The scientists decide to use two different biotechnological strategies in the SAME plant:

• Strategy 1: Introduction of a specific cry gene from Bacillus thuringiensis to confer insect resistance.
• Strategy 2: Use of RNA interference (RNAi) technology to silence a nematode-specific essential gene (cpc-1, encoding a cytochrome P450 essential for nematode survival).

For Strategy 2, the scientists design a construct that, when expressed in the soybean root cells, produces double-stranded RNA (dsRNA) complementary to the nematode's cpc-1 mRNA. When the nematode feeds on root cells, it ingests this dsRNA.

(a) In Strategy 1, the cry gene product exists as an inactive protoxin inside the bacterial cell. Explain the two-step mechanism by which this protoxin becomes lethal to the pod borer larva but does NOT harm humans consuming the crop. [2 marks]

(b) In Strategy 2, trace the molecular pathway from the moment the nematode ingests the dsRNA to the ultimate silencing of the cpc-1 gene. Name the specific enzyme and the intermediate molecule involved. [2 marks]

Accepted Answer

CBSE MARKING SCHEME — 4 Marks Total

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Part (a): Mechanism of Bt toxin — [2 marks]
─────────────────────────────────────────

Step 1 — Solubilisation in alkaline midgut (½ mark):
• The cry gene in soybean cells is expressed as an INACTIVE protoxin (crystalline Bt toxin protein).
• When the pod borer larva ingests plant tissue, the protoxin enters the highly ALKALINE (high pH) midgut of the insect.
• In this alkaline environment, the protoxin is SOLUBILISED (dissolved).

Step 2 — Activation by midgut proteases and mechanism of lethality (1 mark):
• Proteases present in the insect's midgut cleave the protoxin → produce the ACTIVE TOXIN form.
• The active toxin binds to specific receptor proteins on the epithelial cells lining the midgut.
• It creates PORES (ion channels) in the epithelial cell membrane → causes cell swelling and lysis → disrupts midgut integrity → insect dies.

Why it does NOT harm humans (½ mark):
• Human gut is ACIDIC (low pH) — the protoxin is NOT solubilised.
• Even if solubilised, human intestinal cells LACK the specific receptor proteins required for the toxin to bind and form pores.
• Therefore, the protoxin passes through the human digestive system without being activated or binding, posing no harm.

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Part (b): RNAi molecular pathway — [2 marks]
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Step-by-step molecular pathway:

1. INGESTION (¼ mark):
   • The nematode (Heterodera glycines) feeds on soybean root cells and ingests the double-stranded RNA (dsRNA) complementary to cpc-1 mRNA.

2. DICER ACTION — enzyme named (½ mark):
   • Inside the nematode cells, the enzyme DICER (an RNase III-type endonuclease) recognises and cleaves the ingested long dsRNA.
   • Dicer cuts the dsRNA into short fragments of ~21–23 nucleotide pairs called siRNA (small interfering RNA) — the key intermediate molecule.

3. RISC FORMATION (½ mark):
   • The siRNA is incorporated into a multi-protein complex called RISC (RNA-Induced Silencing Complex).
   • The double-stranded siRNA is unwound within RISC; the antisense (guide) strand is retained.

4. TARGET mRNA DEGRADATION → gene silencing (¾ mark):
   • The antisense siRNA strand within RISC guides the complex to the complementary cpc-1 mRNA of the nematode (by Watson-Crick base pairing).
   • RISC cleaves the cpc-1 mRNA → the mRNA is degraded.
   • Result: cpc-1 mRNA is no longer translated → cytochrome P450 enzyme is NOT produced → the nematode CANNOT survive → nematode dies or fails to establish infection in soybean roots.

Summary pathway (to write as a flowchart in answer book):

dsRNA (ingested by nematode)
          ↓  [DICER — endonuclease]
        siRNA (~21–23 bp) — intermediate
          ↓  [incorporated into RISC complex]
   RISC–siRNA complex
          ↓  [base pairs with cpc-1 mRNA]
   cpc-1 mRNA cleaved and degraded
          ↓
   No Cytochrome P450 produced
          ↓
   Nematode death / cpc-1 gene silenced

─────────────────────────────────────────
VALUE POINTS SUMMARY (Examiner Reference)
─────────────────────────────────────────
(a) ½ + 1 + ½ = 2 marks
  • Alkaline pH solubilises protoxin — ½
  • Midgut proteases activate toxin → pores in epithelial cells → lysis — 1
  • Humans: acidic pH + no specific receptors — ½

(b) ½ + ½ + ¾ + ¼ = 2 marks
  • Dicer (enzyme named correctly) — ½
  • siRNA (intermediate named correctly) — ½
  • RISC formation + antisense strand guides complex — ½
  • cpc-1 mRNA degraded → no protein → nematode silenced — ½
  (Award full 2 marks if all four elements: dsRNA → Dicer → siRNA → RISC → mRNA degradation are correctly traced with enzyme and intermediate named)

Question 2

Read the following passage and answer the questions that follow:

A pharmaceutical company is developing a therapeutic protein to treat patients suffering from Type 1 Diabetes Mellitus. The scientists isolate the human insulin gene from pancreatic beta cells. However, when they directly clone the human insulin gene (with introns) into a bacterial expression system (E. coli), the bacteria fail to produce functional insulin protein. The team then adopts an alternative molecular approach using mRNA isolated from beta cells to generate the gene of interest. The final product (pro-insulin) is produced in the bioreactor, purified, and the C-peptide is removed to yield active insulin.

(a) Identify and explain the alternative molecular approach used by the scientists to generate a functional insulin gene suitable for expression in bacteria. Why does this approach solve the problem? [2 marks]

(b) The company produces insulin in large quantities using a stirred-tank bioreactor. State any TWO structural features of a stirred-tank bioreactor that ensure optimal conditions for insulin production, and mention the function of each feature. [2 marks]

Accepted Answer

MARKING SCHEME — 4 Marks

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PART (a) — 2 Marks
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

▶ Alternative approach: cDNA synthesis (using Reverse Transcriptase) [½ mark]

▶ Steps of the approach:
  • mRNA isolated from pancreatic beta cells (which actively transcribe the insulin gene) is used as a template.
  • The enzyme Reverse Transcriptase synthesises a complementary single-stranded cDNA from the mRNA template.
  • DNA Polymerase then converts the single-stranded cDNA into double-stranded cDNA.
  • This double-stranded cDNA is then inserted into the bacterial expression vector. [½ mark]

▶ Why this solves the problem: [1 mark]
  • Bacteria (prokaryotes) LACK the splicing machinery (spliceosomes) needed to remove introns from pre-mRNA.
  • The genomic human insulin gene contains introns (non-coding sequences). If cloned directly, bacteria cannot process the pre-mRNA transcript, so no functional insulin is produced.
  • cDNA is synthesised from processed mRNA (from which introns have already been removed during splicing in the human cell).
  • Therefore, cDNA contains ONLY exon sequences (NO INTRONS) and can be directly transcribed and translated by bacteria to produce functional insulin protein.

[Award full 2 marks for: name of approach + enzyme (½) + steps (½) + clear explanation of no introns in cDNA as reason (1)]

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
PART (b) — 2 Marks
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

Any TWO of the following features with their functions (1 mark each = ½ for feature + ½ for function):

┌─────────────────────┬───────────────────────────────────────────────────────┐
│ Structural Feature  │ Function                                              │
├─────────────────────┼───────────────────────────────────────────────────────┤
│ Stirrer / Agitator  │ Provides uniform mixing of nutrients and oxygen;      │
│                     │ ensures even distribution of cells throughout the     │
│                     │ culture medium for optimal growth.                    │
├─────────────────────┼───────────────────────────────────────────────────────┤
│ Sparger             │ Introduces sterile air/oxygen as fine bubbles into    │
│                     │ the culture medium to maintain aerobic conditions      │
│                     │ required by the microorganisms for insulin synthesis.  │
├─────────────────────┼───────────────────────────────────────────────────────┤
│ Temperature control │ A water jacket / heat exchanger maintains optimal      │
│ jacket              │ temperature for enzyme activity and microbial growth; │
│                     │ prevents overheating due to metabolic activity.       │
├─────────────────────┼───────────────────────────────────────────────────────┤
│ pH sensor / control │ Monitors and maintains optimal pH of the medium so    │
│ system              │ that enzyme activity and cellular processes for        │
│                     │ protein production are not disrupted.                 │
├─────────────────────┼───────────────────────────────────────────────────────┤
│ Foam breaker /      │ Prevents excessive foaming during aeration and        │
│ Antifoam system     │ agitation, which can otherwise damage cells and       │
│                     │ reduce yield.                                         │
└─────────────────────┴───────────────────────────────────────────────────────┘

[Award 1 mark per correct feature–function pair; any 2 correct pairs = 2 marks]

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TOTAL: 4 Marks
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Additional Note (examiner guidance):
• In part (a), if a student writes only 'reverse transcription' without explaining the intron-removal advantage, award maximum 1 mark.
• In part (b), accept any two valid features with accurate functions; do not penalise for diagram if question does not ask for one.

Question 3

Read the following passage and answer the questions that follow:

A biotechnology research team at an agricultural institute was working to develop insect-resistant brinjal (Bti brinjal). They isolated a specific cry gene from a soil bacterium and introduced it into brinjal plants using a naturally occurring soil pathogen as a vector. The transformed plants expressed a protoxin in their tissues. When the target insect pest (Leucinodes orbonalis — shoot and fruit borer) fed on these plants, the protoxin was activated inside the insect's gut, leading to the death of the pest. However, the researchers noticed that the same protoxin had NO toxic effect on mammals, birds, or beneficial insects such as honeybees.

(a) Identify the soil bacterium from which the cry gene was isolated, and name the soil pathogen used as a vector to introduce this gene into brinjal. (1 mark)

(b) Explain the molecular mechanism by which the activated toxin kills the insect pest but is harmless to mammals and birds. (2 marks)

(c) The researchers wanted to confirm that the cry gene had been successfully integrated and expressed in the transgenic brinjal plant. Suggest ONE molecular technique to detect gene integration in the plant genome and ONE technique to confirm that the protein product is being produced. Give a reason for your choice in each case. (1 mark)

Accepted Answer

CBSE MARKING SCHEME — 4 Marks

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(a) [1 mark]

• Soil bacterium (source of cry gene): Bacillus thuringiensis   [½ mark]
• Vector used for gene transfer into brinjal: Agrobacterium tumefaciens (Ti plasmid — disarmed, tumour-inducing genes removed)   [½ mark]

─────────────────────────────────────────
(b) [2 marks]

Step-by-step mechanism:

1. The cry gene in Bacillus thuringiensis encodes an inactive protein called a PROTOXIN (Cry protein / Bt toxin as protoxin).   [½ mark]

2. When the insect pest ingests the plant material, the ALKALINE pH of the insect midgut (pH ~9–10) dissolves and activates the protoxin, converting it into the ACTIVE TOXIN form.   [½ mark]

3. The active toxin binds to specific receptor proteins on the epithelial cells of the insect midgut → creates pores in the cell membrane → cells swell and lyse → midgut epithelium is destroyed → insect stops feeding and dies.   [½ mark]

4. Why it is harmless to mammals and birds:
   • The mammalian/avian gut is ACIDIC (stomach pH ~2), so the protoxin is NOT activated.
   • Mammals lack the specific receptor on gut epithelial cells to which the activated toxin binds.
   • Therefore, the toxin is digested as a simple protein and causes no harm.   [½ mark]

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(c) [1 mark]

Technique to detect GENE INTEGRATION in plant genome:
→ Southern Blotting (or PCR / DNA fingerprinting)   [¼ mark]
Reason: Plant genomic DNA is digested with restriction enzyme, electrophoresed, transferred to membrane, and probed with a labelled cry gene probe. A positive hybridisation signal confirms that the cry gene sequence has integrated into the plant chromosome.   [¼ mark]

Technique to confirm PROTEIN EXPRESSION:
→ ELISA (Enzyme Linked Immunosorbent Assay) OR Western Blotting   [¼ mark]
Reason: ELISA uses a specific antibody raised against the Bt Cry protein; an enzyme-linked colour reaction confirms that the Cry protoxin is being translated and is present in the plant tissue extract. (Western blotting similarly detects the protein by antibody after gel separation.)   [¼ mark]

─────────────────────────────────────────
VALUE-POINT SUMMARY FOR EXAMINER:

(a) Bacillus thuringiensis + Agrobacterium tumefaciens (Ti plasmid) — 1 mark (½ each)
(b) protoxin → alkaline gut activation (½) + receptor binding → pore formation → cell lysis (½) + mammals: acidic gut / no specific receptor → not activated (½+½) — 2 marks
(c) Gene integration: Southern blot / PCR + reason (½); Protein detection: ELISA / Western blot + reason (½) — 1 mark

Total = 4 marks
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Question 4

Read the following passage carefully and answer the questions that follow:

A team of agricultural scientists in India wanted to develop a crop plant that could resist attacks by a devastating stem borer insect, which was causing massive yield losses. They isolated a specific gene from a soil bacterium known to produce a protein toxic to certain insects. This protein exists as an inactive form inside the bacterial cell but gets converted to its active form in the alkaline pH of the insect's midgut, causing cell lysis and ultimately death of the insect. The scientists successfully introduced this gene into the crop plant using Agrobacterium tumefaciens as a vector. Field trials showed that the transgenic plants were highly resistant to stem borer but had no adverse effect on mammals, birds, or beneficial insects such as bees.

(a) Identify the soil bacterium from which the insecticidal gene was isolated. Name the specific type of gene involved and write the full form of the abbreviated gene name.

(b) Explain the molecular mechanism by which the protein encoded by this gene kills the insect pest but does NOT harm mammals.

(c) The scientists used Agrobacterium tumefaciens as a vector. Name the specific plasmid present in this bacterium used for gene transfer. What modification must be done to this plasmid before it can be used as a cloning vector?

(d) Suggest ONE advantage and ONE limitation of using such insect-resistant transgenic crops over conventional chemical pesticides.

Accepted Answer

MARKING SCHEME — Biotechnology and its Applications (4 marks)

─────────────────────────────────────────
(a) Identification of bacterium and gene [1 mark]
─────────────────────────────────────────
• Soil bacterium: Bacillus thuringiensis (Bt)
• Gene involved: cry gene (crystalline gene)
  Full form: 'cry' stands for 'crystal' — these genes encode Cry proteins (crystalline insecticidal proteins / δ-endotoxins)
(Award 1 mark for correctly identifying Bacillus thuringiensis AND naming the cry gene with its full form)

─────────────────────────────────────────
(b) Molecular mechanism of insecticidal action and safety to mammals [1 mark]
─────────────────────────────────────────
• The cry gene encodes the Bt toxin, which is produced inside the bacterial cell (or transgenic plant cell) as an INACTIVE PROTOXIN (pro-toxin / inactive form).
• When the insect larva (stem borer) feeds on the transgenic plant, the protoxin is ingested and enters the insect's midgut.
• The alkaline pH of the insect midgut (approximately pH 9–10) solubilises and cleaves the protoxin into its ACTIVE TOXIN form.
• The active toxin binds to specific receptor proteins on the epithelial cells lining the midgut wall.
• This binding creates PORES in the midgut epithelial cell membrane, causing the cells to swell and lyse, ultimately destroying the insect's gut and leading to the death of the insect.

Why it does NOT harm mammals:
• The mammalian digestive system has an ACIDIC pH (stomach pH ~1–2), so the inactive protoxin cannot be solubilised or converted into its active form.
• Additionally, mammals lack the specific midgut receptor proteins to which the active toxin binds.
• Hence, Bt toxin remains inactive and harmless in the mammalian gut.
(Award 1 mark for correctly explaining: inactive protoxin → activated by alkaline pH of insect midgut → pore formation → cell lysis → insect death; AND stating that acidic pH of mammalian gut / absence of specific receptors prevents activation/action)

─────────────────────────────────────────
(c) Ti plasmid and required modification [1 mark]
─────────────────────────────────────────
• The specific plasmid present in Agrobacterium tumefaciens used for gene transfer is the Ti plasmid (Tumour-inducing plasmid).
• The Ti plasmid contains a segment called T-DNA (Transferred DNA) that naturally integrates into the host plant's genome and causes crown gall disease (tumour formation).
• Modification required: The Ti plasmid must be DISARMED — i.e., the tumour-inducing genes present on the T-DNA must be removed/inactivated so that the plasmid can no longer cause tumour formation in the host plant, while retaining its ability to deliver and integrate foreign DNA into the plant genome. The gene of interest (cry gene) is then inserted into this disarmed Ti plasmid for transfer.
(Award 1 mark for correctly naming the Ti plasmid AND stating that it must be disarmed by removing/inactivating its tumour-causing genes before use as a cloning vector)

─────────────────────────────────────────
(d) One advantage and one limitation of insect-resistant transgenic crops over conventional chemical pesticides [1 mark]
─────────────────────────────────────────
Advantage (any one):
• Reduces the need for chemical pesticide application, thereby lowering production costs for farmers and reducing chemical residues in the environment (soil, water, food).
• Environmentally safer — the Bt toxin is biodegradable, highly specific to the target pest, and does not persist in the ecosystem as chemical pesticides do.
• Protects beneficial insects (e.g., bees, natural predators) that are harmed by broad-spectrum chemical pesticides.
• Cost-effective for farmers as repeated pesticide spraying is not required.

Limitation (any one):
• Prolonged exposure may lead to development of resistance in the target insect population (e.g., stem borers may evolve resistance to Bt toxin over time).
• Risk of unintended gene flow to wild relatives of the crop through cross-pollination, potentially creating 'superweeds' or disrupting natural ecosystems.
• May have unforeseen long-term effects on non-target organisms or soil microbiota.
• Raises regulatory, biosafety, and ethical concerns regarding release of genetically modified organisms into the environment.
(Award 1 mark for stating at least ONE valid advantage AND ONE valid limitation; both must be present for the mark to be awarded)

Question 5

Assertion (A): A bacterium transformed with a recombinant plasmid in which the foreign gene has been inserted within the tetracycline-resistance gene (tet^R) of pBR322 will grow on ampicillin-containing medium but NOT on tetracycline-containing medium.
Reason (R): Insertion of foreign DNA into tet^R by insertional inactivation disrupts the gene, so the bacterium loses tetracycline resistance while retaining ampicillin resistance encoded by the intact amp^R gene.

Accepted Answer

Correct option: (A) — Both A and R are true, and R is the correct explanation of A.

Explanation (for examiner reference):
• pBR322 carries two selectable markers: amp^R (ampicillin resistance) and tet^R (tetracycline resistance).
• When foreign DNA is ligated into the BamHI/SalI/ClaI site located WITHIN tet^R, the tet^R gene is physically disrupted (insertional inactivation) → protein product of tet^R is non-functional → bacterium becomes tetracycline-sensitive.
• The amp^R gene is unaffected → bacterium retains ampicillin resistance.
• Replica-plating strategy: colonies are first grown on amp plates (all transformants survive) → replica-plated onto tet plates → colonies that FAIL to grow on tet plates are the recombinants (insertional inactivation of tet^R confirmed).
• Colonies that grow on BOTH amp and tet plates carry the self-ligated (non-recombinant) plasmid — tet^R intact.
• R correctly and completely explains the phenotype described in A, so option (A) is correct.

Question 6

Which of the following is the CORRECT match between a transgenic organism and the product/gene it is associated with?

Accepted Answer

Correct answer: (A) Rosie (transgenic cow) — Human alpha-lactalbumin in milk

Reasoning (for examiner reference):
• Rosie was the first transgenic cow (1997); the human alpha-lactalbumin gene was inserted into her genome → her milk contained human alpha-lactalbumin, making it nutritionally more balanced for human infants.

Why the other options are INCORRECT:
• (B) Bt cotton carries cry I Ac gene (not cry II Ab) for resistance against bollworm (Helicoverpa). Cry II Ab is used against bollworm in Bt brinjal context and other insects — NOT corn borer in cotton.
• (C) Golden Rice contains the psy gene (from daffodil/Narcissus) for β-carotene (provitamin A) synthesis — NOT human insulin.
• (D) AAT sheep produce alpha-1-antitrypsin (AAT) in their milk (used to treat emphysema) — NOT beta-galactosidase (which is the product of the lac Z gene in lac operon).

∴ Option (A) is the only fully correct match.

Question 7

Assertion (A): In the production of human insulin using recombinant DNA technology, the insulin gene is first obtained as cDNA rather than directly from the human genome.
Reason (R): Bacteria lack the splicing machinery required to remove introns from eukaryotic pre-mRNA, so a cDNA copy (made from processed mRNA) is used to ensure the correct protein is expressed.

Accepted Answer

Correct option: (A) Both A and R are true, and R is the correct explanation of A.

Explanation (for examiner reference):
• Assertion is TRUE: Human insulin cDNA (complementary DNA) is used in recombinant insulin production. cDNA is synthesised from mature mRNA (already spliced) using reverse transcriptase, giving a DNA copy that contains ONLY the coding sequence (exons) with NO introns.
• Reason is TRUE and correctly explains A: Bacteria (e.g., E. coli — the expression host for recombinant insulin) are prokaryotes and possess NO spliceosome machinery. They cannot remove introns from a eukaryotic genomic DNA copy. Therefore, if genomic DNA were cloned directly, the bacteria would transcribe an mRNA containing introns and produce a non-functional or truncated protein. Using intron-free cDNA overcomes this problem entirely.
• Since R directly and completely explains WHY cDNA (not genomic DNA) is used, option (A) is correct.

Key NCERT terminology:
— cDNA: complementary DNA synthesised from mature mRNA by reverse transcriptase.
— No introns in cDNA → can be expressed directly in prokaryotic host.
— Humulin: brand name of recombinant human insulin (Eli Lilly, 1982) — first approved rDNA pharmaceutical.

Question 8

A biotechnology company produces recombinant human insulin by inserting the insulin gene into *E. coli*. However, the insulin produced is initially non-functional. Which of the following is the most likely reason for this, and what additional step is required to obtain functional insulin?

Accepted Answer

Correct answer: (B)

Explanation (for examiner reference / 1-mark award):
• Recombinant human insulin (Humulin) is produced by inserting the A-chain gene and B-chain gene separately into two different *E. coli* strains.
• Each strain produces one chain independently.
• The A and B chains are extracted and then combined in vitro under controlled conditions so that the correct disulphide bonds form between them, yielding biologically active (functional) insulin.
• This is the additional step required — it cannot occur automatically inside *E. coli* because the bacterium lacks the eukaryotic post-translational processing machinery that normally excises the C-peptide from proinsulin and forms the correct inter-chain disulphide bonds.

Why the other options are wrong:
• (A) — Incorrect: *E. coli* does have its own RNA polymerase and can transcribe genes; the problem is not at the transcription level.
• (C) — Incorrect: cDNA is indeed used (so no introns), but this is already accounted for in the standard rDNA procedure; the non-functional nature of the product is not due to introns at this stage.
• (D) — Incorrect: The genetic code is universal — prokaryotes and eukaryotes use the same codons; *E. coli* ribosomes can translate human mRNA sequences.

Mark: 1 mark for choosing (B). No partial credit for MCQ.

Question 9

A farmer in Punjab notices that after introducing Bt-cotton in his fields, he has significantly reduced his pesticide expenditure compared to his neighbour who grows non-Bt cotton. He consults an agricultural scientist who explains the molecular basis of this resistance. Which of the following correctly explains why Bt-cotton plants are able to kill the bollworm larvae that feed on them?

Accepted Answer

Correct answer: (B)

The cry1Ac gene from Bacillus thuringiensis is expressed in Bt-cotton plants, producing an inactive protoxin (Bt toxin).

Mechanism:
• The bollworm larva ingests the plant tissue containing the protoxin.
• The alkaline pH of the insect's midgut (gut lumen) converts the inactive protoxin into its active toxic form.
• The activated toxin binds to specific receptor proteins on the epithelial cells lining the midgut.
• This binding creates pores in the epithelial cell membrane → cells swell and lyse → the midgut is destroyed → larva dies.

Why other options are wrong:
• (A) is incorrect because the Bt toxin is NOT active in all environments — it is specifically activated only in the alkaline pH of the insect midgut (it remains inactive in the slightly acidic/neutral pH of mammals, making it safe for humans).
• (C) is incorrect because the cry1Ac protein is produced inside the plant cells and acts when the larva ingests the plant tissue — it is not secreted into the soil.
• (D) is incorrect because Bt toxin works through pore formation (protein toxin mechanism), NOT through RNA interference (RNAi).

Question 10

A farmer in Punjab plants Bt cotton to protect his crop from bollworm attack. His neighbour asks: 'Which organism is the original source of the gene that makes Bt cotton resistant to bollworm?' The correct answer is:

Accepted Answer

Correct Answer: (B) Bacillus thuringiensis — a soil bacterium whose cry gene encodes the insecticidal protein.

Value point (1 mark): The cry gene responsible for Bt toxin (insecticidal crystal protein) in Bt cotton is derived from the soil bacterium Bacillus thuringiensis.

Question 11

Name the two antibiotic resistance genes present in plasmid pBR322. How is the method of 'insertional inactivation' used to identify recombinant colonies?

Accepted Answer

The two antibiotic resistance genes in pBR322 are:
• ampR (ampicillin resistance gene)
• tetR (tetracycline resistance gene)

Insertional inactivation:
• If a foreign DNA fragment is inserted into the tetR gene (at a restriction site within it), the tetR gene is disrupted and no longer functional.
• Recombinant bacteria: grow on ampicillin plates (ampR intact) but do NOT grow on tetracycline plates (tetR inactivated) → identified as recombinants.
• Non-recombinant bacteria: grow on BOTH ampicillin and tetracycline plates (both genes intact).

[½ mark: ampR; ½ mark: tetR; ½ mark: recombinant colonies fail to grow on tetracycline; ½ mark: non-recombinants grow on both — Total 2 marks]

Question 12

A farmer in Punjab grew a high-yielding variety of wheat that his family had cultivated and selected over generations. A multinational company isolated a gene responsible for disease resistance from this wheat, incorporated it into a new commercial variety, obtained a patent in a foreign country, and started selling seeds at high prices — without compensating the farming community. (i) What term is used for this type of unauthorised commercial exploitation of biological resources/traditional knowledge? (ii) Name ONE legal mechanism India has established to prevent such exploitation of its traditional biological knowledge.

Accepted Answer

(i) The term used for this unauthorised commercial exploitation of biological resources and associated traditional knowledge without proper authorisation or compensation to the original country/community is **Biopiracy**. (1 mark)

(ii) India has established the **Traditional Knowledge Digital Library (TKDL)** — a digital database that documents India's traditional knowledge (including medicinal plants, agricultural varieties, and folk practices) in a format accessible to international patent offices, so that prior art can be cited to reject fraudulent patents. (1 mark)

[Award 1 mark for correctly naming 'Biopiracy'; 1 mark for naming TKDL or mentioning the Biological Diversity Act, 2002 as the legal framework — either is acceptable. Full NCERT context: other examples of biopiracy include the Basmati rice patent (RiceTec Inc., USA, 1997) and the turmeric wound-healing patent — both were successfully challenged.]

**Total: 2 marks**

Question 13

A biotechnology company wants to produce human insulin using bacteria. They decide to use cDNA made from insulin mRNA rather than the actual human insulin gene isolated directly from chromosomal DNA. Give TWO reasons why cDNA is preferred over genomic DNA for expression of human insulin in bacteria.

Accepted Answer

Award 1 mark for each correct reason (any two):

(1) cDNA has NO INTRONS (non-coding intervening sequences) — it is made from processed mRNA which has already had introns removed by splicing. Bacteria LACK the splicing machinery (spliceosome) to remove introns, so genomic DNA would produce a non-functional protein in bacteria. [1 mark]

(2) cDNA is a smaller, compact sequence (only exons) — it is easier to clone into an expression vector and the bacterial cell can directly translate the mRNA produced from it into a functional insulin protein. [1 mark]

[Note to examiner: Accept any two of the above points. The key biological principle is: prokaryotes (bacteria) cannot process eukaryotic pre-mRNA / remove introns, so cDNA (which mirrors mature mRNA) must be used for expression of human genes in bacterial systems.]

Question 14

Mention the steps involved in the preparation of cDNA from a eukaryotic mRNA. Why is cDNA preferred over genomic DNA when expressing a eukaryotic gene in a bacterial host?

Accepted Answer

Steps of cDNA preparation: (1) mRNA is used as a template with the enzyme reverse transcriptase → single-stranded cDNA is synthesised. (½ mark) (2) The mRNA strand is degraded (by RNase H) and DNA polymerase synthesises the complementary strand → double-stranded cDNA is formed. (½ mark)

Reason for preferring cDNA in bacteria: cDNA has NO INTRONS (non-coding sequences), because it is made from processed/mature mRNA from which introns have already been spliced out. (½ mark) Bacteria lack the RNA splicing machinery to remove introns from pre-mRNA; therefore, if genomic DNA (which contains introns) were used, bacteria could not produce the correct functional protein. cDNA ensures the entire sequence is coding (exons only) and can be directly transcribed and translated in the bacterial host. (½ mark)

[Total: 2 marks — ½ + ½ + ½ + ½]

Question 15

A biotechnology company is producing human insulin (Humulin) on a large scale using recombinant E. coli. The scientists used complementary DNA (cDNA) instead of the genomic DNA of the insulin gene for expression in bacteria. They also grew the recombinant bacteria in a stirred-tank bioreactor before downstream processing.

(i) Why did the scientists prefer cDNA over genomic DNA for expressing the human insulin gene in E. coli? (1 mark)
(ii) Name any TWO essential structural components of a stirred-tank bioreactor that ensure optimum conditions for bacterial growth and production of insulin, and state the function of each. (2 marks)

Accepted Answer

MARKING SCHEME (Total: 3 marks)

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Part (i) — 1 mark
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

cDNA is preferred over genomic DNA because:

• cDNA is synthesised from mature mRNA (processed mRNA) using reverse transcriptase and therefore contains NO INTRONS (non-coding intervening sequences). [½ mark]

• E. coli (a prokaryote) LACKS the cellular machinery (spliceosome) to remove introns from pre-mRNA. If genomic DNA (which contains introns) were used, the introns would NOT be spliced out and a non-functional / truncated protein would be produced. [½ mark]

► Hence cDNA ensures the complete, correct insulin protein is expressed in the bacterial host.

(Award full 1 mark for any answer that clearly states: cDNA has no introns AND bacteria cannot remove introns.)

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
Part (ii) — 2 marks (½ mark per component name + ½ mark per function = 4 × ½ = 2 marks)
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━

Any TWO of the following components with their correct functions:

┌─────────────────────────┬───────────────────────────────────────────────────┐
│ Component               │ Function                                          │
├─────────────────────────┼───────────────────────────────────────────────────┤
│ Stirrer / Impeller      │ Provides continuous agitation/mixing of the       │
│                         │ culture medium to ensure uniform distribution of  │
│                         │ nutrients and O₂ throughout the vessel.           │
├─────────────────────────┼───────────────────────────────────────────────────┤
│ Sparger                 │ Introduces sterile air (O₂) into the medium as    │
│                         │ fine bubbles to meet the aerobic requirements of  │
│                         │ the growing bacteria.                             │
├─────────────────────────┼───────────────────────────────────────────────────┤
│ Temperature control     │ Maintains optimum temperature for enzyme activity │
│ jacket / Water jacket   │ and bacterial growth; prevents denaturation of    │
│                         │ the product (insulin).                            │
├─────────────────────────┼───────────────────────────────────────────────────┤
│ pH sensor / pH control  │ Monitors and maintains the optimum pH of the      │
│ system                  │ medium to ensure maximum enzyme activity and      │
│                         │ bacterial growth.                                 │
├─────────────────────────┼───────────────────────────────────────────────────┤
│ Foam control /          │ Prevents excessive foaming during agitation which │
│ Antifoam system         │ could damage cells and reduce productivity.       │
└─────────────────────────┴───────────────────────────────────────────────────┘

(Award ½ mark for correct name of component + ½ mark for correct function, for each of the TWO components chosen.)

━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
VALUE POINT SUMMARY
━━━━━━━━━━━━━━━━━━━━━━━━━━━━━━
• (i): cDNA has no introns / bacteria cannot splice introns → 1 mark
• (ii): Any 2 components correctly named with function → 2 × 1 mark

TOTAL = 3 marks

Question 16

With reference to recombinant DNA technology, explain the role of the following in cloning a foreign gene into a bacterial host: (i) Restriction endonuclease, (ii) DNA ligase, (iii) Selectable marker. Give one example of a selectable marker used in the vector pBR322.

Accepted Answer

**Answer (CBSE Marking Scheme Style — 3 marks)**

**(i) Restriction endonuclease (Molecular scissors): [1 mark]**
- Recognises a specific **palindromic sequence** in the DNA and cuts both strands at defined positions, producing **sticky ends** (short, single-stranded overhangs).
- The same restriction enzyme is used to cut both the **vector DNA** and the **foreign/insert DNA**, so that complementary sticky ends are generated on both — enabling them to join.
- *Example:* EcoRI recognises 5′-GAATTC-3′ and cuts between G and A, producing 4-nucleotide 5′ sticky ends (5′-AATT overhang).

**(ii) DNA ligase (Molecular glue / Biological needle and thread): [1 mark]**
- After the insert DNA (with sticky ends) anneals to the complementary sticky ends of the vector, **DNA ligase** forms covalent phosphodiester bonds, permanently joining the insert to the vector.
- This creates a stable **recombinant DNA molecule** (chimeric DNA / rDNA).

**(iii) Selectable marker + example in pBR322: [1 mark]**
- A **selectable marker** is a gene present on the vector that helps in **identifying and selecting transformed host cells** (cells that have taken up the recombinant DNA) from non-transformed cells.
- It works by conferring a detectable phenotype (e.g., antibiotic resistance or fluorescence) to the transformed cells.
- *In pBR322:* The two selectable markers are **amp^R** (ampicillin resistance gene) and **tet^R** (tetracycline resistance gene).
 - **Insertional inactivation** is used: if a foreign DNA is inserted into the *tet^R* gene, this gene is disrupted → the recombinant clone grows on ampicillin plates (because *amp^R* is intact) but **fails to grow on tetracycline plates** → identified as a recombinant transformant.
 - Non-transformants fail to grow on both plates (no antibiotic resistance at all).

**[Any one correct example of a selectable marker in pBR322 = amp^R or tet^R is sufficient for the mark]**

Question 17

What is gene therapy? Describe the steps involved in the gene therapy carried out to treat ADA (Adenosine Deaminase) deficiency in humans. Why is this treatment considered only a partial cure?

Accepted Answer

**Gene Therapy — Definition (½ mark)**
Gene therapy is a technique of introducing a functional/normal gene into the cells of an individual to correct a genetic disorder caused by a defective or absent gene.

**ADA Deficiency Background (½ mark)**
- ADA (Adenosine Deaminase) deficiency: autosomal recessive disorder; absence of the enzyme ADA severely impairs immune function (T-lymphocytes are non-functional).
- First successful gene therapy was performed in 1990 on a 4-year-old girl.

**Steps in ADA Gene Therapy (1½ marks — ½ mark each for any 3 steps)**
1. Lymphocytes are withdrawn from the patient's blood.
2. A functional ADA cDNA (complementary DNA, without introns) is introduced into the lymphocytes using a retroviral vector (retrovirus as vehicle).
3. The genetically engineered (ADA gene-containing) lymphocytes are multiplied in culture in the laboratory.
4. These activated lymphocytes are then infused back into the patient's body, where they produce functional ADA enzyme.

**Why it is only a partial / temporary cure (½ mark)**
- The genetically engineered lymphocytes are somatic cells (not stem cells) and are not immortal — they have a limited lifespan.
- As these cells die, new infusions of engineered lymphocytes are required periodically.
- For a permanent cure, the ADA gene must be introduced into the bone marrow stem cells (early embryonic cells) so that all daughter lymphocytes permanently carry and express the functional ADA gene.

**[Award marks as: Definition = ½ | Background/first case = ½ | Steps (any 3) = 1½ | Partial cure reason = ½ | Total = 3 marks]**

Question 18

A biotechnology company has developed a genetically modified cotton variety that is resistant to bollworm attack. A farmer notices that his neighbouring farmer's non-GM cotton crop is severely damaged by bollworms, while the GM cotton crop is completely unaffected. The farmer asks a biotechnology student to explain: (i) Name the microorganism and the specific gene responsible for this pest resistance. (ii) How does the protein encoded by this gene kill the bollworm? (iii) Why is this protein harmless to humans who consume cotton seed oil?

Accepted Answer

(i) Microorganism and gene: (1 mark)
• Microorganism: Bacillus thuringiensis (Bt) — a gram-positive soil bacterium.
• Gene: cry gene (e.g., cry1Ac gene for cotton bollworm / Helicoverpa armigera).
[Award ½ mark for correct organism + ½ mark for correct gene name]

(ii) Mechanism of bollworm killing: (1 mark)
• The cry gene encodes a Bt toxin protein that exists as an inactive protoxin (crystalline inclusion body / crystal protein) inside Bacillus thuringiensis.
• When the bollworm larva ingests the plant tissue, the alkaline pH of the insect's midgut activates the protoxin into its active toxic form.
• The activated toxin binds to specific receptor proteins on the epithelial cells of the midgut wall.
• This creates pores in the cell membrane → cells swell and lyse → insect larva dies.
[Award 1 mark for: activation in alkaline pH + pore formation/cell lysis — any two correct steps]

(iii) Harmless to humans: (1 mark)
• The Bt toxin is a protoxin that requires specific alkaline conditions (high pH, as in insect gut) for activation.
• The human digestive system is acidic (stomach pH ≈ 2), so the protoxin is NOT activated.
• Additionally, specific receptor proteins on which the toxin acts are present in insect gut epithelium but are ABSENT in human intestinal cells.
• Therefore, the protein is digested as a normal dietary protein in humans and causes no harm.
[Award 1 mark for any correct reason: acidic human gut / absence of specific receptors / protein digested normally — any one valid reason acceptable]

Question 19

What is gene therapy? Explain the steps involved in gene therapy used to treat ADA (Adenosine Deaminase) deficiency. Why is this treatment considered temporary?

Accepted Answer

**Gene Therapy:** Introduction of a functional/normal gene into the cells of a person with a genetic disorder to correct or compensate for the defective gene. [½ mark]

**ADA Deficiency Background:** ADA (Adenosine Deaminase) deficiency is an autosomal recessive disorder caused by deletion/mutation of the gene encoding adenosine deaminase enzyme — crucial for normal functioning of the immune system. [½ mark]

**Steps in Gene Therapy for ADA Deficiency:** [1½ marks — ½ per step]

1. **Isolation of lymphocytes:** Lymphocytes are withdrawn from the blood of the patient (ADA-deficient child — first successful gene therapy, 1990, in a 4-year-old girl).

2. **Introduction of functional ADA gene:** A functional ADA cDNA (using a retroviral vector) is introduced into the isolated lymphocytes in vitro (in culture).

3. **Reinfusion:** The genetically engineered (ADA-expressing) lymphocytes are then returned/infused back into the patient's body, where they produce functional ADA enzyme and restore immune function.

**Why the treatment is temporary:** [½ mark]
The lymphocytes are somatic (non-dividing/short-lived) cells and do not pass the corrected gene to daughter cells. As these lymphocytes die, the patient requires periodic infusions of freshly engineered lymphocytes. For a permanent cure, the ADA gene would need to be introduced into the bone marrow stem cells (which are dividing cells that can perpetuate the corrected gene).

Question 20

A biotech company wants to protect a commercially important crop (chickpea) from a devastating root-knot nematode (Meloidogyne javanica) without using chemical pesticides. Their scientists propose introducing a construct into chickpea plants that produces double-stranded RNA (dsRNA) complementary to a nematode-specific gene essential for its survival.

(i) Name the molecular mechanism that will be triggered inside the nematode cells when it feeds on the transgenic chickpea roots, and identify the specific enzyme that initiates this pathway. (1 mark)

(ii) Explain the sequence of molecular events that will ultimately prevent the nematode from surviving on the transgenic plant. (1 mark)

(iii) Why is it important that the dsRNA construct is designed to target a gene that is specific ONLY to the nematode and NOT present in the chickpea plant itself? Give one molecular reason and one ecological/ethical reason. (1 mark)

Accepted Answer

MARKING SCHEME — 3 marks total

─────────────────────────────────────
Part (i) [1 mark]
─────────────────────────────────────
• Molecular mechanism triggered: RNA interference (RNAi) / gene silencing. [½ mark]
• Specific enzyme that initiates the pathway: DICER (an endonuclease / RNase III-type enzyme that cleaves dsRNA into short interfering RNAs / siRNAs of ~21–23 nucleotides). [½ mark]

─────────────────────────────────────
Part (ii) [1 mark] — sequence of molecular events
─────────────────────────────────────
Award 1 mark for correctly describing all the following steps in order:

Step 1: Transgenic chickpea root cells produce dsRNA complementary to the target nematode-specific mRNA.

Step 2: When the nematode (Meloidogyne javanica) feeds on transgenic roots, the dsRNA enters its cells.

Step 3: Inside nematode cells, DICER cleaves the dsRNA → short interfering RNA (siRNA) fragments (≈21–23 bp).

Step 4: siRNA strands are incorporated into the RISC complex (RNA-Induced Silencing Complex); the sense strand is discarded.

Step 5: The antisense strand of siRNA within RISC base-pairs with the complementary nematode mRNA → RISC cleaves and degrades the mRNA.

Step 6: The target gene (essential for nematode survival) is silenced → nematode cannot produce the essential protein → nematode development/survival is disrupted → nematode dies or fails to complete its life cycle → crop is protected.

(Award full 1 mark if student covers: dsRNA entry → DICER → siRNA → RISC → mRNA degradation → nematode death. Partial steps = ½ mark.)

─────────────────────────────────────
Part (iii) [1 mark — ½ + ½]
─────────────────────────────────────
• Molecular reason [½ mark]: If the dsRNA sequence is complementary to a chickpea gene, RNAi will also silence that gene within the plant's own cells, potentially disrupting an essential plant function/protein → causing unintended harm to the crop plant itself (auto-silencing / off-target gene silencing in the host).

• Ecological/Ethical reason [½ mark]: If the construct targets a gene conserved across many organisms (e.g., beneficial soil organisms, pollinators, or humans), the dsRNA/siRNA could affect non-target species in the ecosystem — violating the principle of species-specific biocontrol and raising biosafety/GEAC regulatory concerns about release of GMOs that cause ecological harm beyond the intended pest.

(Accept any one valid ecological reason: harm to non-target soil organisms / biodiversity loss / ethical concerns over unapproved release of GMOs / regulatory violation under GEAC guidelines.)

Biotechnology and its Applications: Class 12 Biology Practice Questions

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