Sterilization methods and gene transfer techniques in plant tissue culture MCQs With Answer

Introduction: Sterilization methods and gene transfer techniques are foundational for successful plant tissue culture and genetic manipulation in medicinal plant biotechnology. This quiz compilation offers M.Pharm students a focused set of questions that cover practical and theoretical aspects of surface and media sterilization, control of contamination, and a spectrum of gene transfer methods including Agrobacterium-mediated transformation, particle bombardment, protoplast techniques, and selection strategies. Questions emphasize mechanism, critical parameters, advantages and limitations, and steps to improve transformation efficiency and regeneration. Use these MCQs to consolidate conceptual understanding and laboratory decision-making for designing reliable transformation and aseptic culture experiments.

Q1. Which sterilization method is most appropriate for heat-labile antibiotic solutions used in plant tissue culture media?

• Autoclaving at 121°C for 15 minutes
• Membrane filtration through a 0.22 µm filter
• Dry heat at 160°C for 2 hours
• Treatment with 70% ethanol followed by air-drying

Correct Answer: Membrane filtration through a 0.22 µm filter

Q2. What is the commonly recommended sequence for surface sterilization of small leaf or stem explants to minimize microbial contamination while preserving tissue viability?

• Soak in 0.1% mercuric chloride for 10 minutes, rinse twice in sterile water
• Immerse in 70% ethanol for 30 seconds, then treat with 1–2% sodium hypochlorite for 10–15 minutes, followed by sterile water rinses
• Treat with 2% hydrogen peroxide for 1 hour and then blot dry
• Expose explants to UV light for 20 minutes and then place directly onto medium

Correct Answer: Immerse in 70% ethanol for 30 seconds, then treat with 1–2% sodium hypochlorite for 10–15 minutes, followed by sterile water rinses

Q3. Standard autoclave parameters widely used to sterilize plant culture media are which of the following?

• 100°C at atmospheric pressure for 60 minutes
• 121°C at ~15 psi for 15–20 minutes
• 134°C at 30 psi for 3 minutes
• 80°C for 2 hours in a water bath

Correct Answer: 121°C at ~15 psi for 15–20 minutes

Q4. What is the major drawback of using mercuric chloride (HgCl2) for explant sterilization despite its effectiveness?

• It is ineffective against fungal spores
• It causes substantial environmental hazard and residual toxicity to plant tissues
• It is too expensive for laboratory use
• It evaporates rapidly and cannot be rinsed off

Correct Answer: It causes substantial environmental hazard and residual toxicity to plant tissues

Q5. Which pore size membrane filter is generally recommended to obtain sterile, bacteria-free solutions for plant tissue culture?

• 0.8 µm
• 0.45 µm
• 0.22 µm
• 1.2 µm

Correct Answer: 0.22 µm

Q6. Which chemical is commonly used as an antifungal additive in culture media or during explant pretreatment to reduce fungal contamination?

• Tetracycline
• Carbendazim (Bavistin)
• Kanamycin
• Amphotericin B

Correct Answer: Carbendazim (Bavistin)

Q7. Which sterilization approach is most suitable for decontaminating laboratory rooms and is classified as sporicidal when used as a vapor?

• 70% ethanol wipe-down
• Vaporized hydrogen peroxide (VHP)
• Sodium hypochlorite solution sprayed and left to dry
• Boiling water fogging

Correct Answer: Vaporized hydrogen peroxide (VHP)

Q8. Which compound is commonly added during Agrobacterium co-cultivation to induce vir gene expression and enhance T-DNA transfer efficiency?

• Kanamycin
• Acetosyringone
• Silver nitrate
• Polyethylene glycol (PEG)

Correct Answer: Acetosyringone

Q9. For non-destructive, real-time visualization of transgene expression in living plant tissues which reporter is most often used?

• β-glucuronidase (GUS) assay
• Green fluorescent protein (GFP)
• LacZ (β-galactosidase)
• Neomycin phosphotransferase II (NptII)

Correct Answer: Green fluorescent protein (GFP)

Q10. The floral dip method for transformation is most effective and widely used with which model plant species?

• Triticum aestivum (wheat)
• Zea mays (maize)
• Arabidopsis thaliana
• Oryza sativa (rice)

Correct Answer: Arabidopsis thaliana

Q11. Which of the following is a principal advantage of particle bombardment (biolistic transformation) over Agrobacterium-mediated methods?

• It exclusively targets only dicot plants for transformation
• It avoids host-range limitations and can deliver DNA to organelles such as chloroplasts
• It never causes multiple copy insertions
• It does not require tissue regeneration after transformation

Correct Answer: It avoids host-range limitations and can deliver DNA to organelles such as chloroplasts

Q12. Which gene transfer method requires isolated plant protoplasts and uses chemical-mediated uptake of DNA?

• Agrobacterium-mediated transformation
• Particle bombardment
• PEG-mediated transformation
• Floral dip

Correct Answer: PEG-mediated transformation

Q13. The bar gene is commonly used as a selectable marker in plant transformation because it confers resistance to which herbicide or compound?

• Kanamycin
• Hygromycin
• Phosphinothricin (glufosinate/Basta)
• Glyphosate

Correct Answer: Phosphinothricin (glufosinate/Basta)

Q14. Typical co-cultivation duration of plant explants with Agrobacterium for efficient transformation is usually around:

• 30 minutes
• 2–3 days
• 4–6 weeks
• 10 seconds of vortexing

Correct Answer: 2–3 days

Q15. Vacuum infiltration during Agrobacterium-mediated transformation improves transformation frequency primarily by:

• Stimulating plant cell division
• Removing air from intercellular spaces and allowing bacterial access to internal tissues
• Activating plant defense genes
• Sterilizing the plant surface

Correct Answer: Removing air from intercellular spaces and allowing bacterial access to internal tissues

Q16. Electroporation facilitates DNA uptake in plant protoplasts by:

• Using chemical surfactants to dissolve the cell wall
• Creating transient pores in the plasma membrane via short high-voltage electric pulses
• Binding DNA to gold particles and shooting them into the cell
• Exploiting bacterial conjugation machinery

Correct Answer: Creating transient pores in the plasma membrane via short high-voltage electric pulses

Q17. The Ti (tumor-inducing) plasmid of Agrobacterium tumefaciens is essential in plant genetic engineering primarily because it:

• Encodes an entire plant metabolic pathway
• Mediates transfer and integration of T-DNA into the plant genome
• Produces antibiotic resistance in plants
• Acts as a selectable marker in bacterial cultures only

Correct Answer: Mediates transfer and integration of T-DNA into the plant genome

Q18. Which factor is most responsible for the low transformation efficiency and difficulty in regenerating some medicinal plant species?

• Excessive promoter activity from CaMV 35S
• Genotype-dependent recalcitrance and poor regeneration capacity of explants
• Use of fluorescent reporters like GFP
• Too high concentration of selectable antibiotic

Correct Answer: Genotype-dependent recalcitrance and poor regeneration capacity of explants

Q19. What is the main distinction between transient expression assays and stable transformation in plant biotechnology?

• Transient assays integrate transgene permanently, stable transformation does not
• Transient assays result in short-term expression without genomic integration, while stable transformation involves integration and heritable expression
• Transient assays always use selectable markers, stable transformation never does
• There is no difference; both terms mean the same

Correct Answer: Transient assays result in short-term expression without genomic integration, while stable transformation involves integration and heritable expression

Q20. To minimize chimeric plants after transformation, the best practice is to:

• Use a very low concentration of selectable agent so untransformed cells can survive
• Regenerate plants from single transformed cells or thoroughly selected calli to ensure uniformity
• Only use particle bombardment because it never produces chimeras
• Omit selection altogether and screen regenerants by PCR

Correct Answer: Regenerate plants from single transformed cells or thoroughly selected calli to ensure uniformity

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