In Agrobacterium mediated transformation, which one of the following approaches is more likely to generate transgenic plants with INCOMPLETE transfer of the passenger gene?
Placement of passenger gene towards left border and marker gene towards right border of T-DNA
Agrobacterium Mediated Transformation Process
Agrobacterium tumefaciens is a bacterium commonly used in plant genetic engineering to introduce foreign genes into plant cells. This process is called Agrobacterium-mediated transformation. The bacterium contains a large plasmid called the Tumor-inducing (Ti) plasmid. A specific segment of this plasmid, known as the T-DNA (Transfer DNA), is transferred from the bacterium into the plant cell nucleus and integrated into the plant genome.
T-DNA Transfer Mechanism
The transfer of the T-DNA is a complex process initiated by signals from wounded plant cells. The T-DNA is delimited by two 25 bp repeat sequences called the Left Border (LB) and the Right Border (RB). The transfer process begins at the Right Border (RB) and proceeds towards the Left Border (LB). A single-stranded copy of the T-DNA is generated and then transferred into the plant cell. Because the transfer starts at the RB and moves towards the LB, genes located closer to the RB are transferred first and have a higher probability of being fully transferred compared to genes located closer to the LB.
Impact of Gene Placement on Transfer
When inserting genes into the T-DNA for transformation (like a selection marker gene and a passenger gene, which is the gene of interest), their position relative to the RB and LB is crucial for successful transfer. If the transfer process is interrupted before reaching the LB, any genes located distal to the interruption point (closer to the LB) will not be transferred, leading to incomplete transfer.
Let's analyze the given options based on this understanding:
- Option 1: Placement of selection marker gene towards left border and passenger gene towards right border of T-DNA
In this scenario, the passenger gene is closer to the RB (the starting point of transfer). This means it is more likely to be transferred completely. The marker gene is closer to the LB, so it is more likely to be incompletely transferred or not transferred if the process stops early. This arrangement is generally preferred to ensure the transfer of the passenger gene, often using the marker gene for selection. This option is less likely to cause *incomplete transfer of the passenger gene*. - Option 2: Expression of selection marker gene under constitutive promoter and passenger gene under tissue-specific promoter
The type of promoter controlling gene expression (constitutive or tissue-specific) affects *when and where* the gene is active *after* it has been successfully transferred and integrated into the plant genome. It does not influence the efficiency or completeness of the T-DNA transfer process itself. Therefore, this option is not directly related to the likelihood of incomplete transfer *during transformation*. - Option 3: Placement of passenger gene towards left border and marker gene towards right border of T-DNA
Here, the marker gene is closer to the RB (start), making it more likely to be transferred fully. The passenger gene is closer to the LB (end). Since transfer proceeds from RB to LB, if the process is prematurely terminated, the passenger gene (being closer to the LB) is more likely to be left behind or only partially transferred. This scenario increases the likelihood of *incomplete transfer of the passenger gene*. - Option 4: Expression of both selection marker gene and passenger gene under constitutive promoters
Similar to Option 2, the type of promoters used (constitutive in this case) affects post-transfer gene expression, not the transfer process itself. This does not directly influence the likelihood of incomplete transfer of the passenger gene during transformation.
Based on the directionality of T-DNA transfer (RB to LB), placing the passenger gene closer to the LB makes it more susceptible to incomplete transfer if the transfer process is prematurely terminated.
| Gene Placement | Likelihood of Incomplete Passenger Gene Transfer |
|---|---|
| Passenger gene near RB, Marker gene near LB (Option 1) | Lower likelihood |
| Passenger gene near LB, Marker gene near RB (Option 3) | Higher likelihood |
Therefore, placing the passenger gene towards the left border and the marker gene towards the right border of the T-DNA is more likely to result in transgenic plants with incomplete transfer of the passenger gene.
Important Questions from Transgenic animals and plants
An experiment was performed to introduce a transgenic trait in a crop plant by Agrobacterium-mediated transformation using a transgene construct in which the transgene was expressed using the CaMV 35S promoter. It was observed that expression levels of the transgenic protein were very low in all transgenic plants while transgene mRNA levels were high and variable among different plants. Further, different transgenic lines contained different numbers of the T-DNA insert. The following statements were made to explain the above observation:
A. Variations in the number of T-DNA inserts in different transgenic plants is due to more number of host cells getting infected with the T-DNA
B. Low expression levels of the transgenic protein in all transgenic plants could be due to codon usage variations between the host plant and the heterologous source of the transgene
C. The coding sequence of the transgene contained sequences that destabilized the transgene mRNA
D. Variation in copy number of T-DNA in different transgenic plants is due to variation in the promoter used to express the transgene.
Which one of the following options represents all correct
statements?Following statements were given regarding factors influencing variation in expression levels of transgene in transgenic plants:
A. Difference in restriction enzyme sites within the T-DNA.
B. Difference in copy number of the transgene.
C. Variations in site of integration of the T-DNA within the plant genome.
D. Presence of multiple promoters within the T-DNA region.
Which one of the following options represents a combination of statements that would NOT lead to variations in transgene expression levels in transgenic plants generated using the same T-DNA/binary vector?
Which one of the following statements related to genetic transformation of plants is correct?
The following statements are made regarding developing a transgenic mouse:
A. The transgenic mouse thus born will be a homozygous transgenic animal and can be maintained by crossing with another transgenic animal.
B. The fertilized transgenic eggs are allowed to develop in vitro.
C. The desired gene is preferably microinjected in male pronucleus after sperm entry in oocyte.
D. For best efficiency, the desired gene is always microinjected in the male gametes and then they are allowed to fertilize the female gametes.
E. Blastocyst stage embryos are transferred to the uterus of hormonally prepared mother.
F. The fertilized eggs are collected from specific strain of mouse.
G. The female mouse of specific strain is superovulated, oocytes are collected and allowed to fertilize in vitro.
Choose the combination of statements arranged in the correct sequence for developing transgenic mouse.
Which one of the following genomes is most appropriate to determine hybrids in plants and animals?
