Given below are a few approaches/techniques used in the analysis of plants: A. Phenotype linked to the marker gene. B. PCR using primers specific to the host genome. C. Southern hybridization D. PCR using transgene-specific primers. Which one of the following combination of approaches/techniques can be used to differentiate between transgenic and non-transgenic plants of a particular variety if the site of insertion is unknown?
A, C and D
Differentiating Transgenic Plants
To distinguish between transgenic and non-transgenic plants of the same variety, especially when the location where the foreign DNA (transgene) inserted into the plant's genome is unknown, we need methods that can detect the presence or effect of this specific foreign DNA.
Let's examine the given approaches:
Phenotype Linked to Marker Gene
Many genetic engineering experiments include a "marker gene" along with the desired gene of interest. This marker gene often provides an easily observable trait (phenotype), such as resistance to a specific herbicide or antibiotic, or production of a colored or fluorescent protein. If the marker gene is successfully inserted and expressed, the transgenic plant will show this specific phenotype, while the non-transgenic plant of the same variety will not. This difference in phenotype allows for straightforward identification of transgenic plants, regardless of where the gene inserted.
- Effectiveness: Yes, this is an effective method if a functional marker gene is used and the phenotype is clearly distinguishable.
PCR using Host Genome Primers
Polymerase Chain Reaction (PCR) using primers designed to amplify a sequence that is naturally present in the plant's original genome will produce an amplification product from both transgenic and non-transgenic plants of the same variety. This is because their core genomes are the same. This method confirms the presence of the host plant DNA but does not provide any information about the presence or absence of the inserted transgene.
- Effectiveness: No, this method cannot differentiate between transgenic and non-transgenic plants as it detects host DNA common to both.
Southern Hybridization Technique
Southern hybridization is a molecular technique used to detect the presence of a specific DNA sequence within a DNA sample. Genomic DNA from the plants is isolated, cut into fragments using restriction enzymes, separated by size using gel electrophoresis, and then transferred to a membrane. This membrane is then probed with a labeled DNA sequence that matches the transgene. If the transgene is present in the plant's DNA, the probe will bind to it, and a signal will be detected. Non-transgenic plants, lacking the transgene, will not show this signal. Southern hybridization is a reliable method for detecting the presence of a transgene and can even indicate the number of insertion sites and sometimes provide information about the integrity of the insertion, even if the exact location is unknown.
- Effectiveness: Yes, this is a classic and effective method for detecting the presence of a transgene.
PCR using Transgene-Specific Primers
PCR can also be used to specifically detect the presence of the transgene sequence. Primers are designed to amplify a unique sequence that is part of the inserted transgene. When DNA from a transgenic plant is used as a template, the PCR will amplify this specific sequence, resulting in a detectable DNA product. When DNA from a non-transgenic plant is used, this sequence is absent, so no amplification product is formed. This method is highly sensitive and specific for detecting the presence of the transgene itself, irrespective of its insertion site within the genome.
- Effectiveness: Yes, this is a highly effective and widely used method for detecting the presence of a transgene.
Combination for Differentiation
Based on the analysis, approaches A (Phenotype linked to marker gene), C (Southern hybridization), and D (PCR using transgene-specific primers) are all capable of differentiating between transgenic and non-transgenic plants by detecting the presence or expression of the transgene or its associated marker. Approach B (PCR using primers specific to the host genome) cannot make this distinction as it targets host DNA present in both types of plants.
Therefore, a combination of approaches A, C, and D can effectively be used to differentiate between transgenic and non-transgenic plants when the site of insertion is unknown.
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?
