1.Tobacco plants are maintained under sterile conditions on MS medium, without any added growth regulators as shoot cultures in Magenta containers.
Young leaves showed much transformation since they contain actively dividing cells. These growing cells incorporate the Agrobacterium genes into the DNA and when the DNA is being transcribed the incorporated Agrobacterium genes become transcribed too. When the Agrobacterium genes become replicated in high frequency in the actively dividing cells, the genes become more expressed compared to old leaves that are composed of less active dividing cells. The concentration of Agrobacterium suspension also affects the rate at which the GUS genes become expressed (Hooykaas & Schilperoort 1992). From the diagram, regions containing a higher concentration of Agrobacterium suspension, the expression of the gene is higher. This indicates that more transfer of the GUS genes occurs in regions that have more CFU. Therefore, high concentration along with the age of the leaf determine the rate at which the GUS genes will be expressed. Young leaves have higher cell division, can take up more Agrobacterium
2.Remove 2-3 healthy leaves with forceps and a scalpel and place in a sterile petri dish with a small amount of sterile water. Leaf explants will be prepared by cutting the leaf into small squares about 5 mm in diameter (LEAF PREP).
It is necessary to consider all possible outcomes that might result. While conducting a transformation experiment, we would wish for all the cells to be transformed. Unfortunately, this is impossible because of various factors involved in the transformation process. Control treatments are placed to ensure that only one element is being tested during the transformation process (Hooykaas & Schilperoort 1992). The wild type while on the antibiotic medium kills the Agrobacterium cells indicating negative results while when on the normal medium it grows indicating positive results. In our experiment, the explants that have not been exposed to Agrobacterium or biolistic DNA treatment is used as the control elements. From this, we expect that the leaves exposed to the experimental treatment should show positive transformation while leaves serving as control factors should show negative factors.
3.LEAF PREP: Pipette 10 ml of the exponential phase culture of Agrobacterium into a petri dish. Using a sterile scalpel on the sterile petri dish provided, cut two of the least damaged leaves into about six horizontal strips (about 5 mm diameter) in a second petri dish. The strips from the base of the leaf usually work
Plasmids are circular in shape and are considered extrachromosomal materials. They are usually used in transformational experiments due to the presence of three distinct features. These features are: they usually have the unique origin of replication, they have selectable gene markers which serve to control the experiment, they also have a cloning site where the foreign DNA is placed (Hooykaas & Schilperoort 1992). The success of transformation experiment depends on the design of the vector used. Binary vectors differ from vectors used in biolistic in that; binary vectors are larger (fragmented) enabling them to carry more foreign DNA while vectors employed in Biolistic experiments are much smaller. An example of a binary vector commonly used in generating transgenic plants is the pCAMBIA1305.2.
4.4. With 2 or 3 of the strips, cut them into about 5 mm lengths, so that the result is small squares of leaf tissue about 5 mm long on each side. Place these explants right side of the leaf facing upwards onto 2-3 plates of regeneration medium. Seal with parafilm, label with your name, group, date and Non-Cocultivated, making sure you label on the lid of the plate. These are your regeneration and selection controls.
In the process of generating stable transformants, it is necessary to use culture explants already exposed to bacterium medium containing several additives (Cheng, et al. 1998). These additives serve a key role in the generation, selection, and recombination of transformants other than providing nutrients for the recombined cells. The TDZ provide a medium where the positively transformed cells can regenerate. On the other hand, the Hygromycin provide a medium where cells that have not been transformed can be isolated. This is because when the foreign DNA get transcribed in the plasmid vector, the region that is sensitive to Hygromycin becomes altered therefore produce changes that are not reflected by non-transformed cells. Finally, the presence of Timentin in the medium serves to kill bacteria that have not been transformed (Cheng, et al. 1998). This, therefore, enables only transformed cells to be regenerated in the unique medium that serves to distinguish between the transformed and non-transformed cells.
5.Place the other leaf pieces in the Agrobacterium suspension for 5-10 minutes. Then remove them gently with forceps and blot them for a short period on some sterile tissue paper to remove excess
In the presence of substrate, The GUS reporter genes digest it producing observable changes. On the other hand, the non-lethal GFP reporter genes do not require substrate but require Florence so as to be expressed. Therefore. The presence of these two genes can be identified based on the presence of either Florence or substrate. Chlorophyll solution serves as the Florence solution.
6.Place leaf pieces right side up onto two petri dishes containing regeneration medium, label with you group members’ initials, group number, today’s date and whether the explants were inoculated with the GUS or GFP vector. Then seal with parafilm and incubate in light in the growth room for 48
This experiment is indeed a definitive molecular test. This is because the shoots expressing the recombinant DNA could be independently isolated and cultured by using a special medium containing unique additives such as Trimentin and Hygromycin among others. PCR is a major technique that can be used in identifying transformed cells. Cells that have been transformed could be amplified. In the PCR, special molecular scissors are used to cut the regions tested to be recombined and then amplified in a series of cycles. In cells being transformed, the foreign gene might not be expressed because of several reasons. Therefore, expression of genes can be used to test whether cells have been transformed. This is used by the use of special medium or substrates. Silencing can also be used to detect the presence of transformed and non-transformed cells. Plasmids contains special marker regions that have cells that get expressed or suppressed depending on whether their sequence has been altered (Mi, Kon & Chung-Mo, 2009). In some plasmids, when molecular markers cut these specific regions on the plasmid, the gene become silenced while in some they are expressed. This can, therefore, serve to determine which plant cell have been transformed.
References
Cheng Z-M et al. (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium
tumefaciens in genetic transformation. Plant Cell Reports 17(8) 646-649
Mi Jung Kim, Kon Baek and Chung-Mo Park (2009) Optimization of conditions for transient
Agrobacterium-mediated gene expression assays in Arabidopsis. Plant Cell Reports doi: 10.1007/s00299-009-0717-z
Hooykaas and Schilperoort (1992) Agrobacterium and plant genetic engineering. Plant Molecular
Biology 19: 15-38.
