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Unit 5: Plant Transformation Technology; Direct DNA Transfer Methods - Practice Quiz

BTY540 — Plant Biotechnology 60 Questions
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1 Agrobacterium-mediated gene transfer is often called an indirect method of plant transformation because it:

agrobacterium mediated gene transfer (indirect method) Easy
A. Directly injects naked DNA into the nucleus
B. Uses a bacterium as a natural vector to deliver genes into plant cells
C. Requires a physical device to shoot DNA into cells
D. Uses electric pulses to open pores in the plant cell membrane

2 Which species of Agrobacterium is most commonly used for plant genetic transformation?

agrobacterium mediated gene transfer (indirect method) Easy
A. Escherichia coli
B. Agrobacterium rhizogenes
C. Bacillus thuringiensis
D. Agrobacterium tumefaciens

3 Crown gall disease in plants is characterized by the formation of:

crown gall disease Easy
A. Root rot and wilting
B. White powdery growth on stems
C. Tumor-like galls at the crown region of the plant
D. Yellow spots on the leaves

4 Crown gall disease is caused by which organism?

crown gall disease Easy
A. Tobacco mosaic virus
B. Agrobacterium rhizogenes
C. Fusarium oxysporum
D. Agrobacterium tumefaciens

5 The abbreviation Ti in Ti plasmid stands for:

Ti Plasmid and T-DNA Easy
A. Tissue-invading
B. Transformation-initiation
C. Tumor-inducing
D. Transfer-integration

6 Which part of the Ti plasmid is actually transferred and integrated into the plant genome?

Ti Plasmid and T-DNA Easy
A. Origin of replication
B. T-DNA
C. opine catabolism region
D. vir region

7 The T-DNA region is bordered by short repeat sequences called:

Ti Plasmid and T-DNA Easy
A. Origin and terminus sequences
B. Left and right border sequences
C. Promoter and terminator sequences
D. Start and stop codons

8 The vir region of the Ti plasmid is mainly responsible for:

vir region and vir regulon Easy
A. Replicating the plant chromosome
B. Processing and transfer of T-DNA into the plant cell
C. Coding for tumor structural proteins
D. Producing opines for bacterial nutrition

9 Expression of the vir genes is typically induced by:

vir region and vir regulon Easy
A. Ultraviolet light exposure
B. The presence of antibiotics
C. Phenolic compounds released by wounded plant cells
D. High temperatures alone

10 Which vir gene product acts as the sensor that detects plant phenolic signals?

functions of vir genes Easy
A. VirC1
B. VirE2
C. VirA
D. VirD2

11 Which Vir protein functions as an endonuclease that nicks the T-DNA borders?

functions of vir genes Easy
A. VirB
B. VirA
C. VirG
D. VirD2

12 The single-stranded T-DNA that is transferred to the plant is called the:

functions of vir genes Easy
A. V-strand
B. M-strand
C. T-strand
D. R-strand

13 Once inside the plant cell, the T-DNA is ultimately integrated into the:

T-DNA transfer and integration of T-DNA in plant genome Easy
A. Chloroplast DNA only
B. Nuclear genome of the plant
C. Cell wall matrix
D. Cytoplasmic membrane

14 Which protein protects the single-stranded T-DNA by coating it during transfer?

T-DNA transfer and integration of T-DNA in plant genome Easy
A. VirD1
B. VirE2
C. VirG
D. VirA

15 In Agrobacterium-mediated transformation, the co-cultivation step involves:

plant co-culture and in-planta transformation Easy
A. Growing two different plant species in the same medium
B. Fusing two plant protoplasts together
C. Incubating plant explants together with Agrobacterium to allow gene transfer
D. Culturing bacteria on antibiotic plates before transfer

16 The floral dip method is an example of which type of transformation?

plant co-culture and in-planta transformation Easy
A. Particle bombardment
B. Microinjection
C. In-planta transformation
D. Protoplast fusion

17 In the chemical method of direct DNA transfer, which compound is commonly used to promote DNA uptake by protoplasts?

Direct DNA transfer methods: chemical methods and electroporation Easy
A. Acetosyringone
B. Ethidium bromide
C. Polyethylene glycol (PEG)
D. Agarose

18 Electroporation transfers DNA into cells by:

Direct DNA transfer methods: chemical methods and electroporation Easy
A. Applying brief electric pulses that create temporary pores in the membrane
B. Coating DNA in lipid vesicles
C. Injecting DNA with a fine glass needle
D. Shooting DNA-coated gold particles into cells

19 In the particle gun (biolistic) method, DNA is usually coated onto microscopic particles made of:

particle gun method Easy
A. Carbon or silica
B. Silver or copper
C. Gold or tungsten
D. Aluminium or zinc

20 Lipofection introduces DNA into cells using:

lipofection Easy
A. Electric shocks to the cell
B. A microscopic glass needle
C. High-speed metal particles
D. Lipid vesicles (liposomes) that fuse with the cell membrane

21 A researcher wants to introduce a herbicide-resistance gene into a dicot plant using Agrobacterium tumefaciens. Why is this method classified as an indirect gene transfer method?

agrobacterium mediated gene transfer (indirect method) Medium
A. The DNA integrates into the chloroplast genome instead of the nucleus
B. The transformation requires an electric pulse to open membrane pores
C. The foreign gene is delivered into the plant cell through a biological vector rather than physical or chemical means
D. The gene is first expressed in bacteria and only the protein is transferred

22 During Agrobacterium infection, wounded plant cells release phenolic compounds such as acetosyringone. What is the primary role of these compounds?

study of mechanism of agrobacterium infection Medium
A. They neutralize plant defense proteins to allow bacterial entry
B. They directly cut the T-DNA at the border sequences
C. They provide a carbon source that the bacterium metabolizes for energy
D. They activate the vir genes by triggering the VirA/VirG two-component signal system

23 Crown gall tumors continue to grow in vitro on medium lacking auxins and cytokinins. What best explains this hormone-independent growth?

crown gall disease Medium
A. The bacterium continuously supplies growth hormones to the tissue
B. The tumor cells lose the need for any hormones due to mutation
C. Opines produced by the cells substitute for plant hormones
D. T-DNA genes cause the plant cells to synthesize their own auxins and cytokinins

24 When constructing a plant expression vector, the native oncogenes within the T-DNA are removed to make it 'disarmed'. What is the main consequence of this modification?

Ti Plasmid and T-DNA Medium
A. The T-DNA can still be transferred and integrated but no longer induces tumors
B. The T-DNA loses its border sequences and cannot be transferred
C. The plant cells fail to produce opines but form larger galls
D. The Ti plasmid can no longer replicate inside Agrobacterium

25 Which sequences on the T-DNA are absolutely essential for its transfer into the plant genome, and what is a key feature of them?

Ti Plasmid and T-DNA Medium
A. The opine synthesis genes located at both ends of the T-DNA
B. The 25 bp left and right border repeats, with the right border being more critical
C. The onc genes, which mark the start and end of transfer
D. The ori region flanking the T-DNA on both sides

26 The vir region genes are located on the Ti plasmid but outside the T-DNA. What functional advantage does this arrangement provide in a binary vector system?

vir region and vir regulon Medium
A. The vir genes integrate into the plant genome to maintain gene expression
B. The vir genes are co-transferred with the T-DNA into the plant
C. The vir genes prevent the T-DNA from leaving the bacterium prematurely
D. The vir genes can act in trans to mobilize a separate T-DNA on another plasmid

27 VirD2 forms a covalent attachment to the 5' end of the single-stranded T-strand. What is the significance of this attachment during T-DNA transfer?

functions of vir genes Medium
A. It pilots the T-strand into the plant nucleus and protects the 5' end
B. It cleaves the T-DNA at both border sequences repeatedly
C. It phosphorylates VirG to keep the vir regulon active
D. It coats the entire length of the T-strand to prevent degradation

28 Which pair of vir gene products is chiefly responsible for generating the single-stranded T-DNA by nicking at the border repeats?

functions of vir genes Medium
A. VirA and VirG
B. VirB and VirD4
C. VirE1 and VirE2
D. VirD1 and VirD2

29 VirE2 protein coats the T-strand as it enters the plant cell. If VirE2 function were lost, what outcome would you most likely expect?

functions of vir genes Medium
A. The vir regulon would remain permanently switched off
B. The T-strand would be more susceptible to degradation and inefficiently imported into the nucleus
C. Opine catabolism in the bacterium would be blocked
D. The T-DNA would never be excised from the Ti plasmid

30 The VirB proteins and VirD4 together perform which specific function in Agrobacterium-mediated transformation?

functions of vir genes Medium
A. Form the type IV secretion channel that exports the T-complex into the plant cell
B. Sense phenolic compounds released from wounded plant tissue
C. Integrate the T-DNA into the plant chromosome
D. Nick the T-DNA borders to release the T-strand

31 Integration of T-DNA into the plant genome is generally described as occurring at which type of site?

T-DNA transfer and integration of T-DNA in plant genome Medium
A. Exclusively within the chloroplast genome
B. At fixed centromeric locations on each chromosome
C. Random or quasi-random sites, often in transcriptionally active regions
D. Only at specific homologous sequences matching the T-DNA

32 In the floral-dip (in-planta) transformation of Arabidopsis, transformed progeny are recovered without a tissue-culture regeneration step. Why is this method advantageous?

plant co-culture and in-planta transformation Medium
A. It removes the need for any selectable marker in the T-DNA
B. It avoids somaclonal variation and the labor of in-vitro regeneration
C. It guarantees a single-copy insertion in every transformant
D. It transforms the chloroplast genome directly for stable expression

33 During the co-cultivation step, Agrobacterium is incubated with plant explants for about 2–3 days before adding antibiotics like cefotaxime. What is the purpose of adding the antibiotic afterward?

plant co-culture and in-planta transformation Medium
A. To select only the plant cells that received the T-DNA
B. To induce the vir genes for better T-DNA transfer
C. To eliminate the Agrobacterium while allowing transformed plant cells to survive
D. To promote shoot regeneration from the explants

34 Polyethylene glycol (PEG)-mediated transformation is typically performed on protoplasts rather than intact plant cells. What is the main reason?

Direct DNA transfer methods: chemical methods and electroporation Medium
A. PEG degrades the plant cell wall enzymatically during the process
B. PEG can only bind to chloroplast membranes inside intact cells
C. Intact cells cannot regenerate after PEG treatment
D. The cell wall blocks DNA uptake, so it must be removed to expose the plasma membrane

35 In electroporation, a brief high-voltage pulse is applied to protoplasts suspended with DNA. What does the pulse accomplish?

Direct DNA transfer methods: chemical methods and electroporation Medium
A. It charges the DNA so it is attracted to the nucleus
B. It creates transient pores in the plasma membrane through which DNA enters
C. It permanently fuses the DNA to the cell membrane
D. It cuts the DNA into fragments small enough to diffuse in

36 The biolistic (particle gun) method is often preferred over Agrobacterium for transforming cereals like maize and rice. Which feature makes it especially suitable for these plants?

particle gun method Medium
A. It exclusively transforms the nuclear genome and never organelles
B. It always produces single-copy, clean insertions
C. It requires no selectable marker for recovery of transformants
D. It can deliver DNA into cells regardless of host-range limitations of Agrobacterium

37 In the biolistic method, DNA is coated onto microprojectiles. Which materials are most commonly used for these particles and why?

particle gun method Medium
A. Aluminium or copper, because they conduct electricity into the cell
B. Iron or steel, because they can be guided by magnetic fields
C. Gold or tungsten, because they are dense, inert, and small enough to penetrate cells
D. Silica or glass beads, because they dissolve after entering the cell

38 Lipofection introduces DNA into cells using cationic liposomes. What property of these liposomes facilitates DNA delivery?

lipofection Medium
A. Their negative charge repels DNA into the cell interior
B. They form covalent bonds with the DNA phosphate backbone
C. They enzymatically digest the cell wall to admit DNA
D. Their positive charge binds negatively charged DNA and fuses with the cell membrane

39 Microinjection allows direct delivery of DNA into a single plant cell or protoplast. Despite its precision, why is it not used for large-scale plant transformation?

microinjection and macroinjection Medium
A. It is highly labour-intensive and low-throughput, treating one cell at a time
B. It cannot deliver DNA into the nucleus accurately
C. It requires the presence of active vir genes to succeed
D. It only works on bacterial cells, not plant cells

40 Macroinjection differs from microinjection primarily in that it:

microinjection and macroinjection Medium
A. Delivers DNA using an electric field instead of a needle
B. Injects DNA into a multicellular region such as developing floral tillers rather than a single cell
C. Uses a finer needle to target individual organelles
D. Requires protoplasts stripped of their cell walls

41 During Agrobacterium infection, the virA gene product perceives phenolic compounds like acetosyringone. However, sugar molecules released from wounded cells enhance vir induction indirectly. Which mechanism best explains the role of monosaccharides in this signaling cascade?

study of mechanism of agrobacterium infection Hard
A. Sugars methylate the virA promoter, permanently activating the two-component system
B. Sugars directly phosphorylate VirG, bypassing the need for VirA autophosphorylation
C. Sugars bind the periplasmic protein ChvE, which then interacts with VirA to increase its sensitivity to phenolics at lower pH
D. Sugars are transported into the bacterium and act as the actual inducers, with phenolics being dispensable

42 The VirA/VirG two-component system regulates the vir regulon. If a mutant Agrobacterium strain constitutively expresses a VirG protein locked in its phosphorylated (active) conformation, what would be the most likely phenotype regarding vir gene expression?

vir region and vir regulon Hard
A. vir expression only when both acetosyringone and low pH are present
B. Complete loss of vir gene expression regardless of inducer presence
C. Constitutive expression of vir genes even in the absence of acetosyringone
D. Expression restricted to virA and virG but not the downstream operons

43 VirD2 and VirE2 are both associated with the T-strand during transfer. What is the critical functional distinction between the nuclear localization signals (NLS) they carry?

functions of vir genes Hard
A. VirE2 is covalently linked to the 5' end while VirD2 non-covalently coats the strand
B. VirD2 has a single C-terminal NLS covalently attached to the 5' end, while VirE2 has bipartite NLSs that coat the entire single strand
C. Both carry identical monopartite NLSs recognized by the same importin
D. Neither carries an NLS; nuclear import depends solely on host VIP1 protein

44 In an octopine-type Ti plasmid, the T-region can be split into TL and TR. In binary vector systems used for transformation, which feature makes the disarmed T-DNA still functional despite removing oncogenes?

Ti Plasmid and T-DNA Hard
A. The opine synthesis genes substitute for the missing border repeats
B. The oncogenes are replaced by vir genes so transfer becomes autonomous
C. The border repeats (25 bp) are retained, and vir genes are supplied in trans from a separate helper plasmid
D. The T-DNA is integrated into the bacterial chromosome to remain stable

45 T-DNA integration into the plant genome occurs predominantly via which mechanism, and what consequence does this have for insertion patterns?

T-DNA transfer and integration of T-DNA in plant genome Hard
A. Site-specific recombination mediated by a Cre-like recombinase encoded by virC
B. Non-homologous end joining (illegitimate recombination), leading to largely random integration sites often with microhomologies at junctions
C. Homologous recombination targeting specific chromosomal loci with high site specificity
D. Rolling-circle replication that produces tandem head-to-tail arrays at centromeres

46 Crown gall tumors continue to proliferate in vitro even after the Agrobacterium is eliminated with antibiotics. Which molecular basis best explains this hormone-autonomous growth?

crown gall disease Hard
A. The bacteria leave behind stable exogenous auxin that persists indefinitely
B. Antibiotics induce endogenous plant tumor suppressor inactivation
C. Opines act as growth hormones stimulating uncontrolled division
D. Integrated T-DNA oncogenes (iaaM, iaaH, ipt) autonomously synthesize auxin and cytokinin in plant cells

47 In biolistic transformation, the choice between gold and tungsten microcarriers involves a trade-off. Why is gold often preferred over tungsten despite its higher cost for certain sensitive cell types?

particle gun method Hard
A. Tungsten cannot bind DNA, so gold is the only viable carrier
B. Gold is chemically inert and non-toxic, whereas tungsten can catalyze DNA degradation and is cytotoxic to some cells
C. Gold particles are larger, ensuring deeper penetration into all cell layers
D. Gold particles are magnetic, allowing precise targeting inside the tissue

48 In protoplast electroporation, increasing the field strength beyond an optimum reduces transformation efficiency. What is the primary reason for this decline at very high field strengths?

Direct DNA transfer methods: chemical methods and electroporation Hard
A. The field neutralizes the negative charge on DNA, preventing uptake
B. Pores close too rapidly to allow DNA entry
C. DNA becomes denatured by the electric field and cannot integrate
D. Irreversible membrane breakdown causes protoplast lysis and cell death

49 The virD2 gene product acts as a relaxase/endonuclease. If a point mutation abolishes only the tyrosine residue at the VirD2 active site while leaving the NLS intact, what is the expected outcome?

functions of vir genes Hard
A. T-strand transfer proceeds normally because VirD1 compensates fully
B. T-strand nicking and covalent 5' attachment fail, so no T-strand is generated for transfer
C. Only the right border is nicked while the left border processing is blocked
D. T-strand is produced but cannot enter the nucleus due to lost NLS function

50 Microinjection into plant cells is technically more challenging than in animal cells. Which structural feature is the principal obstacle, and how is it commonly addressed?

microinjection and macroinjection Hard
A. The presence of multiple nuclei makes single-target injection impossible
B. The rigid cell wall and turgor pressure require protoplasts or immobilization of cells in agarose/agar for injection
C. Chloroplast autofluorescence blocks visualization of the injection needle
D. The absence of a nucleus in mature plant cells prevents targeting

51 The right border (RB) is more critical than the left border (LB) for T-DNA transfer. Which observation and mechanism best support this asymmetry?

T-DNA transfer and integration of T-DNA in plant genome Hard
A. VirD2 remains attached to the 5' end at the RB and defines the leading end of transfer, so RB deletion abolishes transfer while LB deletion is more tolerated
B. The LB contains the origin of replication essential for T-strand synthesis
C. The RB encodes the overdrive-independent promoter for oncogenes
D. Both borders are functionally identical; the asymmetry is purely an artifact of sequencing

52 Lipofection uses cationic liposomes to deliver nucleic acids. Why is the net positive charge of the lipid–DNA complex (lipoplex) mechanistically important for delivery into protoplasts?

lipofection Hard
A. The positive charge promotes association with the negatively charged membrane and facilitates fusion/endocytosis
B. The positive charge neutralizes nucleases in the cytoplasm before uptake
C. It activates the vir regulon to trigger active DNA import
D. It causes the DNA to condense into crystals that pass through channels

53 Historically Agrobacterium was considered a dicot-specific pathogen, limiting monocot transformation. Which combination of factors was most critical in successfully extending the method to cereals like rice?

agrobacterium mediated gene transfer (indirect method) Hard
A. Use of super-virulent strains, addition of acetosyringone during co-cultivation, and choice of highly regenerable target tissues (e.g., immature embryos)
B. Removal of all vir genes and reliance on host recombinases in monocots
C. Elimination of the border repeats to allow whole-plasmid integration
D. Switching from binary vectors to cointegrate vectors exclusively

54 The VirB proteins and VirD4 together form a Type IV secretion system (T4SS). What is being exported through this apparatus during infection?

functions of vir genes Hard
A. The entire Ti plasmid as a double-stranded circle
B. Only opine molecules synthesized in the plant back into the bacterium
C. Acetosyringone to amplify the induction signal in neighbouring bacteria
D. The VirD2–T-strand complex along with effector proteins such as VirE2 and VirF into the plant cell

55 The Arabidopsis floral-dip (in-planta) transformation method bypasses tissue culture regeneration. Which biological target is actually being transformed to yield transgenic progeny, and how is this confirmed?

plant co-culture and in-planta transformation Hard
A. The root meristem, confirmed by staining the dipped shoot
B. The female gametophyte (developing ovule/egg), confirmed by selecting transformed T1 seed progeny rather than the dipped plant
C. The pollen grains only, confirmed by staining anthers
D. The somatic leaf cells, confirmed by callus formation

56 PEG-mediated transformation of protoplasts is a classic chemical method. What is the primary role of polyethylene glycol (PEG) in facilitating DNA uptake?

Direct DNA transfer methods: chemical methods and electroporation Hard
A. It replicates the introduced DNA to increase copy number
B. It covalently links DNA to membrane proteins for active transport
C. It digests the cell wall to create protoplasts before transfer
D. It causes membrane destabilization and reversible permeabilization, promoting endocytosis-like uptake of DNA

57 The 'overdrive' sequence enhances T-DNA transfer efficiency. Where is it located and which Vir proteins interact with it?

Ti Plasmid and T-DNA Hard
A. Just outside the right border; it is bound by VirC1 (and VirC2) to stimulate T-strand production
B. In the middle of the T-DNA; bound by VirE2 during coating
C. At the origin of replication; bound by VirG to initiate transcription
D. Within the left border repeat; bound by VirD2 to terminate transfer

58 Biolistics is the preferred method for chloroplast (plastid) transformation, unlike Agrobacterium. What property of plastid transformation is uniquely enabled here and what mechanism drives stable integration?

particle gun method Hard
A. Integration into the nuclear genome followed by protein targeting to plastids
B. Site-specific integration via homologous recombination into the polyploid plastome, enabling homoplasmic, high-level expression
C. Random NHEJ integration into plastid DNA, giving position effects
D. Episomal replication without integration, requiring continuous selection

59 Some vir genes (e.g., virA, virG) are expressed at low basal levels even without induction, while others (virB, virD, virE) are strongly inducer-dependent. What is the functional logic of this expression hierarchy?

vir region and vir regulon Hard
A. Basal VirA/VirG must be present to sense the signal and then transcriptionally activate the inducible operons upon induction
B. The inducible operons are expressed first to prime the sensor genes
C. All vir genes require identical induction, and basal expression is an artifact
D. VirB/VirD/VirE regulate VirA/VirG in a negative feedback loop before induction

60 Transgenic plants produced by Agrobacterium generally show fewer copies and simpler integration patterns than those made by biolistics. What mechanistic difference best accounts for this?

T-DNA transfer and integration of T-DNA in plant genome Hard
A. Agrobacterium uses site-specific recombinase enzymes absent in biolistics
B. Agrobacterium physically limits DNA to one molecule per cell
C. Single-stranded, protein-capped T-DNA integrates via defined border-mediated ends, whereas biolistic naked DNA undergoes fragmentation and multimerization before random insertion
D. Biolistic DNA is always methylated, blocking multiple insertions
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