Question 1

Agrobacterium–plant interaction — In octopine-type Ti plasmids, the integrated T-DNA within the plant genome typically occurs as how many segments?

Accepted Answer

Introduction / Context: Agrobacterium tumefaciens transfers a defined region of its Ti plasmid (T-DNA) into plant cells, where it integrates into the nuclear genome and drives tumor (crown gall) formation. Octopine-type Ti plasmids characteristically contain two T-DNA regions. Given Data / Assumptions: Different Ti plasmid types (octopine vs nopaline) have different T-DNA organization. Octopine-type T-DNA is often found as two separate segments (TL and TR). These segments encode oncogenes and opine synthesis genes. Concept / Approach: Recall that “TL” (left) and “TR” (right) segments can integrate and express genes in plant cells. Their presence explains phenotypes and opine profiles in transformed tissues. Step-by-Step Solution: Identify plasmid type: octopine-type.Associate with two T-DNA regions: TL and TR.Select “two segments” as the correct organization. Verification / Alternative check: Molecular mapping and restriction analyses of octopine Ti plasmids reveal separate TL and TR inserts. Why Other Options Are Wrong: a,c,d) Do not reflect canonical octopine-type organization.e) While repeats can occur, the hallmark is two segments. Common Pitfalls: Confusing nopaline and octopine plasmid structures; always check plasmid type. Final Answer: Two segments (TL and TR).

Question 2

Crown gall properties — Once induced by Agrobacterium, what are the in vitro characteristics of crown gall tissue?

Accepted Answer

Introduction / Context: Crown gall results from stable integration of T-DNA into the plant genome. After transformation, the tissue becomes autonomous: it can grow without Agrobacterium and often without exogenous hormones, reflecting the expression of T-DNA oncogenes and opine synthesis genes. Given Data / Assumptions: T-DNA integration is stable and heritable in cultured cells. Opine synthesis and auxin/cytokinin biosynthesis genes alter metabolism and growth control. Eliminating the bacteria does not remove the integrated T-DNA. Concept / Approach: Because the tumor phenotype is encoded by plant-integrated T-DNA, crown gall tissue remains tumorous in culture without the bacterium. This autonomy is a cornerstone of Agrobacterium-mediated transformation evidence. Step-by-Step Solution: Induction: Agrobacterium transfers T-DNA → integration.Post-induction culture: remove bacteria; tissue still grows aberrantly.Conclusion: both independence from bacteria and retention of tumor traits occur. Verification / Alternative check: Opine assays and hormone-free growth of tumor lines confirm persistent T-DNA function. Why Other Options Are Wrong: d) Incorrect; tumor properties do not require persistent bacteria.e) Reversion requires loss/silencing of T-DNA, which is not the default. Common Pitfalls: Assuming antibiotic curing of bacteria will cure the tumor; integrated T-DNA remains. Final Answer: Both (a) and (b).

Question 3

Ti plasmid genetics — Where are the virulence (vir) genes that mediate T-DNA processing and transfer located relative to the T-DNA region?

Accepted Answer

Introduction / Context: Agrobacterium tumefaciens uses vir genes to excise, process, and transfer T-DNA into plant cells. These vir genes are essential for infection but are not themselves transferred to the plant; they reside in a distinct region of the Ti plasmid. Given Data / Assumptions: T-DNA borders delimit the DNA that is transferred. virA/virG and other vir operons encode proteins for sensing, processing, and transfer. Only the T-DNA between border repeats integrates into the plant genome. Concept / Approach: Separate the transfer cargo (T-DNA) from the bacterial machinery (vir region). The vir genes operate in trans to mobilize the T-DNA, remaining in the bacterium. Step-by-Step Solution: Identify vir region location → outside T-DNA.Recognize that only T-DNA integrates into plant DNA.Choose the option reflecting this separation. Verification / Alternative check: Binary vector systems split T-DNA and vir functions onto separate plasmids, proving vir can act in trans. Why Other Options Are Wrong: a) If vir were on T-DNA, they would transfer, but they do not.c,d,e) Contradict known Ti plasmid architecture and function. Common Pitfalls: Confusing border sequences (on T-DNA) with vir genes (outside T-DNA). Final Answer: Outside the T-DNA region on the Ti plasmid (separate vir region).

Question 4

Agrobacterium virulence determinants In plant biotechnology, on which genetic element is the virulence trait of Agrobacterium tumefaciens primarily borne and transmitted to plants?

Accepted Answer

Introduction / Context: Agrobacterium tumefaciens is renowned for its natural ability to transfer DNA into plant cells, causing crown gall disease. The core question tests whether you know where the virulence (disease-causing and DNA-transfer) functions reside and how they underpin modern plant genetic engineering. Given Data / Assumptions: The bacterium possesses a large tumor-inducing (Ti) plasmid. Plant tumors (galls) result after a specific DNA segment integrates into the plant genome. We are asked about the location of virulence determinants that enable DNA processing and transfer. Concept / Approach: Virulence is primarily encoded on the Ti plasmid. Two key functional parts are: (1) T-DNA, the segment that is transferred and integrated into the plant genome, and (2) the vir region, a cluster of virulence genes that sense plant signals, process T-DNA at the borders, and export it through a type IV secretion system into plant cells. While some chromosomal genes help bacterial physiology, the essential transfer machinery and the oncogenic/opine traits are Ti plasmid-encoded. Step-by-Step Solution: Identify the DNA element that carries T-DNA and vir genes: the Ti plasmid.Recall that vir genes (virA, virG, virB, virD, etc.) direct sensing, nicking, and export of T-DNA.Remember that the T-DNA portion integrates into plant chromosomes and expresses oncogenes/opine synthase.Conclude that the virulence trait is borne on the Ti plasmid DNA. Verification / Alternative check: Disarmed binary vector systems separate vir functions (on a helper plasmid) from engineered T-DNA, yet transformation still occurs. This proves vir resides in plasmid-borne modules and does not require bacterial chromosomal relocation. Why Other Options Are Wrong: Chromosomal DNA: contributes to general metabolism; not the main virulence locus. Both equally: Ti plasmid specifically encodes transfer/onco traits. Cryptic plasmid: by definition lacks known function. Bacteriophage genome: not involved in Agrobacterium virulence. Common Pitfalls: Confusing the transferred T-DNA (plant-integrated) with the bacterial vir genes (remain in Agrobacterium). Only T-DNA moves; vir genes act in trans from the bacterium. Final Answer: Tumor-inducing (Ti) plasmid DNA

Question 5

Selection in Agrobacterium-mediated transformation Which selectable marker enzymes are routinely used to recover plant cells transformed by Agrobacterium?

Accepted Answer

Introduction / Context: Selectable markers enable researchers to isolate the relatively few plant cells that acquire and express T-DNA after Agrobacterium infection. Understanding common marker enzymes and antibiotics is fundamental for designing transformation experiments and interpreting regeneration results. Given Data / Assumptions: Agrobacterium delivers T-DNA harboring a selectable marker gene into plant cells. Markers typically confer antibiotic resistance that permits growth on selective media. Different crops and protocols favor different antibiotic/marker combinations. Concept / Approach: Widely used markers include neomycin phosphotransferase II (NPTII; kanamycin/G418 resistance) and hygromycin phosphotransferase (HPT; hygromycin resistance). Streptomycin/spectinomycin resistance cassettes are also used in some systems. The best choice depends on plant sensitivity, background tissue responses, and regulatory considerations. Step-by-Step Solution: List common markers: NPTII (kanamycin), HPT (hygromycin), and other aminoglycoside phosphotransferases.Match to selection media: tissues survive only if the marker is expressed.Note species- and tissue-specific sensitivities to antibiotics.Conclude that any of the listed marker enzymes may be used, depending on context. Verification / Alternative check: Transformation protocols across tobacco, tomato, rice, and Arabidopsis document successful selections using kanamycin or hygromycin; some use streptomycin/spectinomycin in plastid or nuclear selection schemes. Why Other Options Are Wrong: NPTII or HPT alone: each is valid but not exclusive. Streptomycin phosphotransferase alone: valid in some cases but not the only standard. lacZ alone: a reporter, not a robust selectable marker in plants. Common Pitfalls: Using antibiotic levels that either fail to suppress escapes (too low) or kill regenerable transformants (too high). Always empirically determine minimal lethal concentrations for the target tissue. Final Answer: Any of the above, depending on the vector and plant species

Question 6

Octopine-type Ti plasmids For octopine Ti plasmids, the right T-DNA segment (TR) is primarily required for which function?

Accepted Answer

Introduction / Context: Ti plasmids differ in T-DNA organization. In octopine-type plasmids, T-DNA is typically split into two segments, TL and TR, each carrying distinct genetic modules that affect tumor development and opine metabolism. Knowing which segment encodes what is a classic detail tested in plant biotechnology. Given Data / Assumptions: We are dealing with octopine Ti plasmids (not nopaline or agropine types). The T-DNA integrates into plant chromosomes and is expressed there. Different T-DNA segments encode oncogenes versus opine synthase genes. Concept / Approach: In octopine strains, the left segment (TL) carries the oncogenic genes that deregulate auxin and cytokinin synthesis, driving tumor formation. The right segment (TR) typically harbors the octopine synthase gene(s), enabling tumor tissues to synthesize the opine octopine, which Agrobacterium can catabolize. Conjugative transfer functions reside outside the T-DNA on plasmid transfer (tra/trb) regions and vir genes are bacterial and not plant-expressed. Step-by-Step Solution: Differentiate TL (tumor genes) from TR (opine synthase).Recall that octopine synthase expression is a hallmark of octopine-type tumors.Note that conjugation genes are not in T-DNA, and vir genes are not transferred to plants.Select the function linked to TR: opine (octopine) biosynthesis enzymes. Verification / Alternative check: Molecular mapping and tumor metabolite profiling show octopine production driven by T-DNA-encoded octopine synthase, reinforcing that TR contributes opine metabolism rather than tumor initiation per se. Why Other Options Are Wrong: Tumor formation: mainly TL in octopine plasmids. Conjugative transfer: plasmid transfer modules are outside T-DNA. All of these/vir in plant nucleus: contradict T-DNA organization and gene locations. Common Pitfalls: Confusing octopine and nopaline plasmids or assuming both TL and TR are interchangeable for oncogenesis. They have specialized roles. Final Answer: Enzymes for octopine biosynthesis (opine synthase expression)

Question 7

Opines in crown gall tumors Which opine classes can be present in plant crown gall tissues induced by Agrobacterium?

Accepted Answer

Introduction / Context: Opines are unusual amino acid–derived compounds synthesized by T-DNA-transformed plant cells. Their profiles (octopine, nopaline, agropine, and related types) help classify Ti plasmids and confirm successful Agrobacterium-mediated transformation. Recognizing opine diversity is important for diagnostics and historical understanding of Ti plasmid biology. Given Data / Assumptions: Crown gall tissues carry integrated T-DNA expressing opine synthase genes. Different Ti plasmid types encode different opine synthases. Question asks which opines can be present overall, not in a single tumor simultaneously. Concept / Approach: Octopine-type Ti plasmids drive octopine synthesis; nopaline-type produce nopaline; agropine-type produce agropine and sometimes related opines. Hence, across crown gall cases, all these opines are encountered. Each opine type reflects the plasmid carried by the infecting Agrobacterium strain. Step-by-Step Solution: Identify opine classes: octopine, nopaline, agropine.Recall linkage: opine type ↔ Ti plasmid genotype.Note that the question asks inclusively about crown gall tissues in general.Select “All of these.” Verification / Alternative check: Paper chromatography and later HPLC analyses of tumor extracts historically distinguished octopine vs nopaline tumors, establishing the correlation between opine profile and Ti plasmid type. Why Other Options Are Wrong: Single opine choices ignore diversity across strains. Normal plant amino acids: opines are novel conjugates not made by untransformed tissues. Common Pitfalls: Assuming a single tumor makes multiple unrelated opines; typically, the opine profile reflects the infecting plasmid type, not a mixture from all classes. Final Answer: All of these

Question 8

Gene delivery into dicot plants Which technique is classically and most widely used to introduce genes into dicotyledonous plants?

Accepted Answer

Introduction / Context: Different plant groups respond differently to gene delivery methods. Dicotyledonous species, including many horticultural crops, have historically been most amenable to Agrobacterium-mediated transformation using Ti plasmid–based vectors, which remains a staple technique in academia and industry. Given Data / Assumptions: The target plant group is dicots. We compare common gene delivery approaches. We seek the technique with broad, established success in dicots. Concept / Approach: Agrobacterium naturally transfers T-DNA into plant cells; disarmed derivatives and binary vector systems provide efficient, genotype-flexible transformation for many dicots (e.g., tobacco, tomato, soybean). While biolistics and electroporation can work across taxa, Ti plasmid infection is the classic, most widely adopted route for dicots due to high integration efficiency and lower copy insertions. Step-by-Step Solution: Identify method with natural DNA transfer mechanism: Agrobacterium with Ti plasmid.Note dicots’ historical susceptibility and tissue culture compatibility.Compare alternatives: biolistics and electroporation are useful but less universally efficient in dicots.Select Agrobacterium-mediated transformation as the best answer. Verification / Alternative check: Extensive literature and protocols (leaf-disc, cotyledon, hypocotyl methods) document routine success of Agrobacterium in dicots; many commercial traits were introduced via this route. Why Other Options Are Wrong: Electroporation and microinjection: niche or protoplast-dependent; not as broadly routine. Biolistics exclusively: works for many monocots; dicots typically favor Agrobacterium. Protoplast fusion: somatic hybridization, not gene transfer of defined constructs. Common Pitfalls: Assuming monocot breakthroughs (e.g., rice biolistics) generalize as the default for dicots; historical and practical preference remains Agrobacterium. Final Answer: Agrobacterium Ti plasmid–mediated infection (Agrobacterium-mediated transformation)

Question 9

Facts about Agrobacterium tumefaciens Which statement best captures its host range and disease association?

Accepted Answer

Introduction / Context: Agrobacterium tumefaciens is both a plant pathogen and a cornerstone of plant genetic engineering. A clear understanding of its natural host range and the disease it causes helps students connect basic pathology with biotechnology applications. Given Data / Assumptions: Crown gall is the classic disease symptom associated with Agrobacterium. Natural susceptibility varies across plant groups. The question seeks the most accurate, concise statement. Concept / Approach: Agrobacterium induces crown galls mainly on dicotyledonous plants (e.g., rose, grapevine). While engineered methods can extend transformation to some monocots, natural infections are largely dicot-biased. Reports of gymnosperm infection are limited and not routine in nature, making the generalization inaccurate. Therefore, the best answer emphasizes crown gall disease and primary dicot host range. Step-by-Step Solution: Identify disease association: crown gall tumors at wound sites.Recall host preference: primarily dicot angiosperms.Exclude overstated claims about gymnosperms or monocots.Select the statement that includes both disease and correct host range. Verification / Alternative check: Plant pathology references describe classic dicot hosts (e.g., grape, stone fruits). Agricultural disease reports align with dicot susceptibility; monocot infections are rare and typically require lab intervention. Why Other Options Are Wrong: Gymnosperm routine infection: not broadly supported. Exclusive monocot infection: opposite of observed natural host range. No disease: contradicted by well-documented pathology. Algae/fungi hosts: incorrect kingdom. Common Pitfalls: Confusing laboratory transformation capability with natural pathogenesis. Success in engineered monocot transformation does not imply broad natural infection. Final Answer: It causes crown gall disease of plants and primarily infects dicotyledonous angiosperms

Question 10

T-DNA borders of the Ti plasmid On the Ti plasmid, the T-region (T-DNA) is flanked by direct repeat border sequences of what length?

Accepted Answer

Introduction / Context: T-DNA transfer begins with precise endonucleolytic nicks at conserved border repeats. Knowing the length of these repeats is central to vector design, primer placement, and understanding how Vir proteins recognize and process the transfer DNA in Agrobacterium-mediated transformation. Given Data / Assumptions: Border repeats occur at left and right ends of the T-DNA. VirD1/VirD2 nick at or near the right border to initiate transfer. The repeat length is conserved among canonical Ti plasmids. Concept / Approach: The classic border element is a 25 base-pair direct repeat sequence present at both T-DNA ends, with the right border serving as the primary nicking site for generating the single-stranded T-strand. These sequences are retained in most binary vectors to ensure proper processing and transfer. Step-by-Step Solution: Recall that both T-DNA borders are short, conserved repeats.Identify the canonical length used in vector maps and cloning manuals: 25 bp.Select 25 bp as the correct value.Recognize that mutations in borders reduce transfer efficiency, underscoring their functional importance. Verification / Alternative check: Sequence alignments of pTi plasmids and widely used binary vectors (e.g., pBIN19 derivatives) show the 25 bp border sequences flanking the T-DNA cassette. Why Other Options Are Wrong: 12, 20, 30, or 100 bp: do not match the canonical 25 bp border repeat length. Common Pitfalls: Confusing the 25 bp border repeats with longer promoter or terminator sequences in the T-DNA payload; the borders are short and specific. Final Answer: 25 bp

Question 11

vir gene regulation in Agrobacterium Which vir genes are constitutively expressed at a basal level and together control plant-induced activation of the other vir operons?

Accepted Answer

Introduction / Context: Agrobacterium senses wounded plant tissues via phenolic signals and activates a regulatory cascade that turns on DNA transfer functions. The central two-component system virA/virG orchestrates this induction, a concept essential for understanding how transformation efficiency depends on signal perception. Given Data / Assumptions: vir genes reside on the Ti plasmid outside the T-DNA. Phenolics (e.g., acetosyringone) from wounded plants trigger the system. Two-component regulatory logic is used (sensor kinase + response regulator). Concept / Approach: virA encodes a membrane-associated sensor histidine kinase that detects phenolic inducers and phosphorylates virG, the cytoplasmic response regulator. Phosphorylated VirG activates transcription of other vir operons (virB, virC, virD, virE), enabling T-DNA processing and export. virA and virG are expressed constitutively at basal levels so they can respond promptly to plant signals. Step-by-Step Solution: Identify the two-component pair: VirA (sensor) and VirG (regulator).Note basal expression allows rapid induction upon signal detection.Downstream vir operons are activated by phosphorylated VirG.Select virA and virG as the correct answer. Verification / Alternative check: Genetic studies show that virA or virG mutants fail to induce other vir genes even in the presence of acetosyringone, demonstrating their upstream regulatory role. Why Other Options Are Wrong: virB, virC, virD, virE encode transfer/processing proteins but are downstream and not the primary sensors/regulators. virA with virB or virG with virE: mismatched pairs lacking the canonical two-component relationship. Common Pitfalls: Assuming all vir genes are induced equally; instead, virA/virG form the master switch that turns on the rest in response to plant cues. Final Answer: virA and virG

Question 12

Origin of opine synthesis In Agrobacterium-induced crown gall tissues, the capacity to synthesize opines is best described as:

Accepted Answer

Introduction / Context: Opines are signature metabolites produced by transformed plant cells in crown gall tumors. Their presence underpins early diagnostics of transformation and historically guided the classification of Ti plasmids. The question probes whether you understand that opine synthesis is plant-executed yet bacterially dictated. Given Data / Assumptions: T-DNA integrates into the plant genome and is expressed by plant transcriptional machinery. The specific opine produced depends on the opine synthase genes carried on the Ti plasmid. Untransformed plant cells do not synthesize opines. Concept / Approach: After transformation, plant cells gain new genes (within T-DNA) that encode opine synthases. Thus, the biochemical capacity resides in plant cells, but its genetic origin is the bacterium’s plasmid. Consequently, both statements are simultaneously true: the plant gains the trait, and its nature is determined by the bacterium’s Ti plasmid type (octopine, nopaline, agropine). Step-by-Step Solution: Recognize that T-DNA is stably integrated and expressed in plant nuclei.Link opine type to Ti plasmid class.Note that uninfected plants lack opine synthase genes.Select “Both (a) and (b)”. Verification / Alternative check: Tumor tissues maintained in vitro without bacteria continue producing the same opines, proving that plant cells themselves carry and express the opine synthase genes acquired from Agrobacterium. Why Other Options Are Wrong: Only (a) or only (b): each is incomplete alone. Normal plant cells or bacterial-only pathway: contradicts the transformation mechanism and tumor biochemistry. Common Pitfalls: Assuming opine production ceases without bacteria present; once integrated, the plant genome encodes the trait autonomously. Final Answer: Both (a) and (b)

Question 13

T-DNA nicking endonuclease Upon perception of plant signals, Agrobacterium produces an endonuclease that nicks the T-DNA at its borders to initiate transfer. Which vir gene encodes this activity?

Accepted Answer

Introduction / Context: Processing of T-DNA is a critical early step in Agrobacterium-mediated transformation. The endonucleolytic nick at the right border generates the T-strand, which is then coated and delivered into plant cells. Identifying the vir operon responsible for nicking clarifies how the transfer intermediate is formed. Given Data / Assumptions: Plant wound signals induce vir gene expression via VirA/VirG. Nicking occurs at conserved T-DNA borders. Multiple vir operons participate in processing and export. Concept / Approach: The virD operon encodes the endonuclease (VirD1/VirD2 complex) that introduces site-specific nicks at T-DNA borders. VirD2 covalently attaches to the 5′ end of the T-strand and, together with other vir proteins, guides the single-stranded DNA through the type IV secretion system. virB forms the secretion channel; virE encodes a single-stranded DNA-binding protein; virA/virG are regulatory. Step-by-Step Solution: Map functions: virA/virG (regulation), virB (secretion), virC (enhances border utilization), virD (nicking), virE (ssDNA coating).Identify the operon with endonuclease activity: virD.Recall covalent linkage of VirD2 to the T-strand 5′ end.Select “virD”. Verification / Alternative check: Mutations in virD abolish border nicking and T-strand formation, blocking transformation even when other vir genes are intact, confirming its essential role. Why Other Options Are Wrong: virA/virG: regulators, not nucleases. virB: secretion pore components. virC: assists right-border utilization but is not the nuclease. virE: ssDNA protection/transport, not nicking. Common Pitfalls: Attributing nicking to virB or virE because they are abundant; abundance does not equal catalytic function—virD performs the cleavage. Final Answer: virD

Question 14

Agrobacterium-mediated gene transfer methods  In plant biotechnology workflows that use Agrobacterium tumefaciens, the transfer of an ‘‘intermediate’’ cloning vector constructed in Escherichia coli into Agrobacterium cells is most commonly accomplis…

Accepted Answer

Introduction / Context: Agrobacterium tumefaciens is a cornerstone of plant genetic engineering because it naturally transfers T-DNA to plant cells. Before plant transformation, researchers often need to move an intermediate vector (assembled in Escherichia coli) into Agrobacterium. Understanding the classical method used to mobilize these plasmids clarifies historical and practical lab procedures. Given Data / Assumptions: Intermediate vectors are typically mobilizable but not self-transmissible. Agrobacterium accepts plasmids via conjugation if transfer (tra/mob) functions are supplied. Multiple laboratory routes exist today (e.g., electroporation), but the question targets the canonical approach. Concept / Approach: Triparental mating uses three partners: (1) an E. coli donor carrying the intermediate vector, (2) an E. coli helper strain carrying a broad-host-range transfer/helper plasmid that supplies tra/mob functions, and (3) the Agrobacterium recipient. The helper plasmid enables conjugative mobilization of the intermediate vector into Agrobacterium where it can replicate or recombine, enabling subsequent plant transformation. Step-by-Step Solution: Identify that the intermediate vector lacks self-transfer functions.Recognize that tra/mob genes on a helper plasmid can be supplied in trans.Recall that classical mobilization procedure is triparental mating.Select ‘‘Triparental mating’’ as the most appropriate answer. Verification / Alternative check: Many legacy protocols (prevalent before routine electroporation of Agrobacterium) describe mixing donor, helper, and recipient on filters/plates, recovering transconjugants with appropriate antibiotic selection—consistent with triparental mating being the standard historical method. Why Other Options Are Wrong: Transformation of Agrobacterium without specialized methods is inefficient. Biparental mating lacks the helper's tra/mob functions for non-self-transmissible plasmids. Transduction in Agrobacterium is not a routine route for plant vectors. Electroporation is possible but was not the classical mainstay implied by ‘‘intermediate vector’’ mobilization. Common Pitfalls: Confusing modern electroporation convenience with the historically dominant conjugation-based mobilization strategy requiring a helper plasmid. Final Answer: Triparental mating (helper plasmid provides mobilization functions)

Question 15

Agrobacterium virulence genes — requirements for T-DNA processing and transfer  During Agrobacterium-mediated transformation, which vir gene products are required for T-DNA processing and transfer into the plant genome?

Accepted Answer

Introduction / Context: The virulence (vir) genes on the Ti plasmid coordinate sensing of plant signals, processing of T-DNA, pilus formation, coating of the T-strand, and transport into plant cells. Knowing which vir loci participate clarifies the orchestrated nature of the transfer machinery. Given Data / Assumptions: Multiple vir operons exist (virA, virG, virB, virC, virD, virE, etc.). T-DNA transfer needs sensing, nicking/processing, coating, and a transport channel. The listed pairs are all known to contribute key functions. Concept / Approach: virA/virG sense phenolic inducers and activate vir transcription; virD (notably VirD2) nicks the right border to generate the T-strand; virE (VirE2) coats the single-stranded T-DNA; virB forms a type IV secretion system for transfer; virC enhances processing at borders. Therefore, multiple vir modules are essential, making ‘‘All of these’’ correct. Step-by-Step Solution: Map each vir pair to a transfer step.Confirm each pair provides a required function (sensing, processing, coating, channel).Conclude that all listed pairs are necessary participants.Choose ‘‘All of these.’’ Verification / Alternative check: Mutations in any of virA/G, virB, virC, virD, or virE reduce or abolish T-DNA transfer, confirming the necessity of the network. Why Other Options Are Wrong: Each individual pair alone is insufficient; the process needs coordinated action of several vir loci. Common Pitfalls: Overemphasizing a single vir gene (e.g., virD2) and overlooking the requirement for activation and transport modules. Final Answer: All of these

Question 16

Protoplast transformation — agents that stimulate direct DNA uptake  Direct uptake of DNA by plant protoplasts (cells without a wall) can be experimentally stimulated using which of the following reagents?

Accepted Answer

Introduction / Context: Protoplast transformation leverages the absence of a rigid wall to introduce exogenous DNA. The efficiency of direct DNA uptake depends on chemical facilitators that promote membrane fusion or transient permeability. Given Data / Assumptions: PEG is widely used to induce membrane perturbation and DNA uptake. Decanal is unrelated to protoplast transformation chemistry. Luciferin is a bioluminescent substrate used for reporters, not for DNA uptake. Concept / Approach: PEG-mediated transformation mixes protoplasts with purified DNA and PEG in an osmoticum; PEG promotes membrane coalescence and facilitates DNA internalization. Divalent cations may assist, but PEG is the hallmark reagent. Decanal and luciferin do not stimulate uptake in this context. Step-by-Step Solution: Identify reagents known to enhance protoplast DNA uptake.Recall standard PEG-Ca^2+ protocols in plant tissue culture.Eliminate unrelated chemicals (decanal, luciferin).Select PEG as the correct facilitator. Verification / Alternative check: Classic plant transformation protocols consistently employ PEG to achieve reproducible DNA uptake into protoplasts, confirming its role. Why Other Options Are Wrong: Decanal and luciferin serve unrelated functions; ‘‘All of these’’ is incorrect because only PEG promotes uptake here. Calcium chloride alone without PEG is insufficient for high-efficiency plant protoplast transformation. Common Pitfalls: Confusing PEG-mediated chemical transformation with electroporation or liposome-mediated delivery, which are distinct methods. Final Answer: Polyethylene glycol (PEG)

Question 17

Liposome-mediated gene transfer in plants — features of the method  Which statements correctly describe liposome-mediated gene transfer (bioliposome fusion) into plant protoplasts?

Accepted Answer

Introduction / Context: Liposome-mediated delivery packages nucleic acids in phospholipid vesicles that can merge with cellular membranes. In plants, the absence of the cell wall (protoplast preparation) facilitates direct fusion and cytosolic release of DNA or RNA. Given Data / Assumptions: Liposomes are biocompatible carriers for nucleic acids. Membrane fusion is a central step for payload delivery. PEG is often used as a fusogen to increase fusion frequency. Concept / Approach: In liposome-mediated transfer, plasmid DNA is enclosed inside lipid bilayers to shield it and allow membrane fusion. Protoplasts present a plasma membrane accessible for fusion. PEG enhances fusion probability. Therefore, all three listed statements accurately reflect the method, while bacteriophage transduction is unrelated. Step-by-Step Solution: Encapsulate plasmid DNA within liposomes.Mix liposomes with plant protoplasts under fusogenic conditions.Optionally add PEG to increase fusion and delivery efficiency.Regenerate cell walls and select transformants. Verification / Alternative check: Experimental reports show PEG-enhanced liposome fusion increases gene transfer rates, supporting the combined statements. Why Other Options Are Wrong: Phage transduction operates in bacterial hosts and is not a plant protoplast delivery mechanism. Common Pitfalls: Conflating chemical (PEG), physical (electroporation), and vesicular (liposome) delivery methods; they are distinct but can be combined strategically. Final Answer: All of the above

Question 18

Octopine Ti-plasmid T-DNA organization — role of the left segment (TL)  In octopine-type Ti-plasmids, what is the primary function encoded by the left T-DNA segment (TL)?

Accepted Answer

Introduction / Context: Octopine-type Ti plasmids often contain two T-DNA regions, termed TL (left) and TR (right). Their genetic content determines tumor induction and opine synthesis in infected plant tissues. Given Data / Assumptions: TL and TR carry different functional gene sets. Auxin and cytokinin biosynthesis genes drive tumor formation. Opine synthesis genes determine the opine type produced by transformed cells. Concept / Approach: In octopine plasmids, TL typically harbors genes required for phytohormone overproduction leading to uncontrolled cell division (tumor formation). TR frequently encodes the opine synthesis enzymes (e.g., octopine synthase). Conjugation genes are not part of T-DNA; they reside elsewhere on the plasmid. Agropine biosynthesis pertains to agropine-type plasmids, not octopine types. Step-by-Step Solution: Differentiate TL (tumorogenic functions) from TR (opine synthesis).Note that opine class specified in the stem is ‘‘octopine,’’ not ‘‘agropine.’’Exclude conjugation functions as non–T-DNA.Select tumor formation as the TL function. Verification / Alternative check: Molecular maps of octopine Ti plasmids assign auxin/cytokinin genes to TL and octopine synthase to TR, corroborating the selection. Why Other Options Are Wrong: Agropine biosynthesis is associated with a different Ti plasmid type. Conjugative transfer functions are not within T-DNA. ‘‘All of these’’ cannot be true because these functions are distributed differently. Common Pitfalls: Confusing opine class names and assuming all Ti plasmids share identical T-DNA organization. Final Answer: Tumor (crown gall) formation via auxin/cytokinin synthesis genes

Question 19

Nopaline Ti-plasmid — number of integrated T-DNA segments  When integrated into the plant nuclear genome, the nopaline-type Ti-plasmid T-DNA is typically present as how many continuous T-DNA segments?

Accepted Answer

Introduction / Context: Ti plasmids are grouped by opines synthesized in transformed tissue (e.g., nopaline, octopine, agropine). Their T-DNA organization differs, and recognizing these patterns aids vector design and interpretation of transformation outcomes. Given Data / Assumptions: Nopaline-type Ti plasmids commonly have one T-DNA region. Octopine-type plasmids often possess two (TL and TR). The question targets the nopaline configuration only. Concept / Approach: In nopaline Ti plasmids, the T-DNA integrated into the plant genome is typically a single, continuous segment bounded by left and right border sequences. In contrast, octopine types can have two discrete T-DNA regions. Therefore, the correct choice is a single segment. Step-by-Step Solution: Recall opine-based classification of Ti plasmids.Match nopaline type with a single T-DNA region.Select ‘‘A single segment of T-DNA.’’Exclude answers that correspond to octopine features or atypical arrangements. Verification / Alternative check: Comparative Ti plasmid maps consistently depict one T-DNA in nopaline types, validating this answer. Why Other Options Are Wrong: Two segments describe many octopine plasmids. Three or four segments are not canonical. Tandem repeats can occur in some events, but are not the default organization implied by the question. Common Pitfalls: Assuming all Ti plasmids share the same dual-segment architecture; they do not. Final Answer: A single segment of T-DNA

Question 20

Liposome-mediated delivery — stability and protection of nucleic acids  In plant liposome-mediated gene transfer, how do liposomes affect the nucleic acid cargo?

Accepted Answer

Introduction / Context: Liposomes are lipid bilayer vesicles that encapsulate biomolecules, including nucleic acids, shielding them from extracellular degradation and facilitating delivery into cells. In plant systems, this is most effective with protoplasts. Given Data / Assumptions: Nucleases in extracellular milieu can degrade naked DNA/RNA. Encapsulation within liposomes limits nuclease access. Bilayer vesicles provide a stabilizing microenvironment during delivery. Concept / Approach: The protective and stabilizing roles of liposomes are central to their utility. They do not inherently replicate DNA; instead, they carry and release it after fusion or endocytosis. Thus, both protection from nucleases and improved stability are correct statements. Step-by-Step Solution: Identify risks to naked nucleic acids (nuclease degradation).Note how lipid encapsulation reduces exposure.Recognize that vesicles maintain cargo integrity during handling and fusion.Select ‘‘Both (a) and (b).’’ Verification / Alternative check: In vitro digestion assays show reduced nuclease access to encapsulated nucleic acids, confirming protection and improved stability. Why Other Options Are Wrong: Liposomes do not make nucleic acids unstable nor do they replicate DNA; replication requires polymerases, not lipid vesicles. Common Pitfalls: Assuming delivery vehicles alter the genetic material enzymatically; liposomes are carriers, not catalysts. Final Answer: Both (a) and (b)

Gene Transfer in Plants Questions