Agrobacterium Mediated Tobacco Transformation By: Patrick Kudyba Section: 002 April 15, 2015
Introduction Transformation is an important tool to plant biotechnologists because it is an easy way to insert desired genes into a plant s genome. Without this process plant biotechnologist would just be breeders selecting for the right genes, but with transformation they can look to other species for desirable genes to help the plant survive or yield more fruit. Transformation can be done 2 ways, via a gene gun or via agrobacterium. Using a gene gun we coat particles of gold with the gene of interest and then shoot it at plant cells so that they can take it up into their genome. Agrobacterium on the other hand can be used because of its properties in plants. It can inject its DNA into a plant cell and if we give it the right plasmid it can inject the plant cells with our desired gene to be taken up by the pant genome. In this lab we transformed tobacco using agrobacterium. Agrobacterium mediated transformation works by using a clonal vector and marker genes. The agrobacterium naturally insert their DNA into plant using the TDNA on the Ti plasmid. Using this process we can insert our own genes of interest into the Ti plasmid where the TDNA is. We also insert marker genes along with the gene of interest so that we can identify if the gene was inserted properly and is being expressed. The marker genes flank the left side of the gene of interest. This is because of the way that TDNA is inserted into the genome. The TDNA has a right and left border and is inserted right to left. Because of this mechanism the TDNA in its entirety does not always make it into the genome, so they put the gene of interest closer to the right border to ensure that it at least makes it through. Then the kanamycin resistance gene is next and the rest of the marker genes after that. The other 2 marker genes usually used are GUS and GFP genes. They both produce fluorescence so that we can visually see that the genes have been inserted properly and are being expressed. If the plant grows but does not show the fluorescence, it could be due to the fluorescence genes being cut off in the insertion process. These genes are not necessary for the plant to survive and other tests can be done to ensure that they are transgenic so that the process does not have to be done all over again. The next step is to grow the plant up from just transgenic tissue. The way in which this is done is again through the natural response of the plant and bacteria. The result is the production of callus tissue, the plant version of stem cells. These cells can grow and then differentiate into whatever tissue the plant needs. Additionally they form at the site of infection and so they are the most likely to contain the transgenic DNA. These calli are then placed in regeneration media and are allowed to form new plants from just transgenic tissue giving a totally transgenic plant that can then be bred with other transgenic plants to create a transgenic line. This technology is very important in the field of plant biology so that we can use natural systems to insert the genes that we desire into certain plants. This opens up an avenue to modify most plants and design them to include a multitude of different kinds of genes to satisfy a variety of needs.
Materials and Methods Tobacco transformation via agrobacterium is a 5 step process: infection/co-cultivation, regeneration/selection, kanamycin assay, rooting, and observation of gene expression. Each step is vital to the successful transformation of genes into the tobacco plant. Step 1 is infection and co-cultivation. This is the step where the bacterium containing the desired gene in a plasmid will infect the explants and deliver the plasmid to the explants chromosome. The cocultivation will then allow the bacterium enough time to properly infect the explant thoroughly. This is done by scoring and washing the explants with agrobacterium that contain the desired transgene plasmid. The explants are then grown with the agro together on growth media for a week so that the bacteria can infect the explant cells with the transgene plasmid and transform them. Step 2 is transferring the explants to the recovery and selection media. The purpose of this step is to help the plant recover from the infection and to select for cells that contain kanamycin resistance. The media used is recovery media to help the plants regenerate from being infected and begin to produce shoots from calli that have already formed from the co-cultivation. The media is also a selection media that contains kanamycin antibiotic. If the cells are resistant to the antibiotic then the plant contains the transgene and will begin to produce shoots. This selects for only transgenic cells to recover and make a whole plant using just transgenic cell lines. Step 4 is transferring specific shoots from the explants to rooting media. The purpose of this step is to take shoots and to induce rooting so that the plant can become a whole plant, and also as another check for selection. The shoots could either be transgenic or escapes. Escapes are shoots that have escaped selection by not touching the kanamycin media by the plant moving and growing or some other means. Transferring these shoots straight into the rooting media also containing the antibiotic kanamycin will show if they are true transgenic plants or escapes. The transgenic plants will grow and produce roots while the escapes will die because they do not contain the kanamycin resistance gene. Rooting is done by taking shoots from the explants at least 1 cm long with a meristem and at least 2 leaves. The shoots are stuck upright into the rooting media and left to incubate; there should be about 5 shoots per growth container. Step 5 is observation of gene expression by collecting data on the rooting efficiency and the GFP expression in the newly growing plants. This is important to determine how well the rooting procedure was and how many of the viable shoots were actually escapes. Additionally the GFP expression shows whether or not the plant contains and expresses the genes in the transgene addition. The GFP marker is at the end of the addition so if the plant is expressing GFP then it also contains the desired gene of interest; in this case kanamycin resistance. Step 3 is the kanamycin assay, which is there to check the germination rate, ratio of insertion, and the level of expression of GFP the marker gene in the transgene plasmid that was inserted. These checks are all here to determine how well the plant took hold of the plasmid and how well it expresses the genes that are now incorporated into its genome, as well as the amount of insertions based on the ratio of living and dying germinated seeds. This assay is done by taking 50 seeds from the self-pollinated tobacco plant and germinate them on selection media containing kanamycin. The seeds were originally supposed
to be poured over the media but instead we just placed each seed in a grid formation to ensure there was enough space between seeds. The seeds were incubated for a week and then were checked for number of germinated seeds, green vs white shoots, and the green shoots were observed under a florescence microscope to look for the green fluorescence of the GFP expression. Results/Observations Part 1: Infection and co-cultivation Leaf observations were taken before the leaves were scored and washed and after 1 week of cocultivation. 2 leaves were taken to be control and 2 sets of 2 leaves were taken to be transgenic. All leaves looked healthy and green and were taken from the same tobacco plant. After infection the leaves were scored/ cut through across the mid vein but they still looked green and other than the exterior cuts, there was no signs of damage. After a week the control leaves were much larger in diameter, and displayed a lighter green pigment. The midveins were much lighter in color and at the cut marks there were calli forming, most notably around the midvein. On the transgenic explants the leaves also expanded and became much lighter in color, and only small calli formed, mostly just looked like buds. Additionally there was bacteria overgrowth on the leaves and in the media. Part 2: Regeneration and selection After wash, one leaf per set was taken and placed in selection media. Each leaf got its own selection media container. After one week, the control (C) was still a very healthy green color, and 2 huge calli formed at the midvein with shoots beginning to sprout from the top. The shoots had some new leaves growing that were dark green, and new calli forming underneath the explant with some shoots forming and growing down into the media. The first transgenic (T1) began to look sickly turning a yellow/brown color. There are some calli forming, very small but in a large quantity, with some shoots forming from them and producing green leaves. The second transgenic (T2) also turned a sickly green color with a slight white coloration. There were large calli forming at the midvein and stem of the explant. Smaller calli formed under the explant forming shoots under the explant. Also to note, the media was much lower than when the explants were first introduced to the media. After the second week the control looked less healthy as an explant but had a major callus formed in the middle of the explant with dark green leaves sprouting from the top. The explant enlarged again and most of the media is gone at this point. The T1 explant had one large shoot from the midvein however the shoot looks sickly and turning brown. However, there are large green shoots under the explant, with whole leaves forming. The T1 explants as a whole looks more brown and dying. The T2 explant looks to be dying at this point, there are large areas of brown/white, shriveled dead tissue. Underneath the explant there are calli formed with lively green shoots growing down. Total number of shoots per explant Control: 100 x 5 = 500 T1: 40 x 6 = 240 T2: 50 x 8 = 400 Part 4: Rooting
The young green shoots were placed upright in the rooting media for each set. Four shoots were selected from the control, three shoots from the T1 transgenic, and 4 were taken from the T2 transgenic. They were all healthy looking shoots with at least 1 cm of stem. After one week, the control had one out of the 4 shoots rooted. All the shoots looked to be healthy shoots and growing but none other were growing roots. There was some white tissue on one of the non-rooted shoots. The T1 transgenic shoots had one shoot rooted out of the 3 selected. The rooted shoot had some dead leaves but overall looks to be healthy and growing. Of the non-rooted shoots one looks to be dead with all white tissue and the other looks very sickly with shriveled leaves and some brown tissue. On the T2 transgenic, 3 out of the 4 shoots rooted, 2 were substantial roots with some lateral movement. Those 2 shoots were very green and had very large leaves. One shoot budded a root but is turning whit and no growing very much. The shoot that did not root is turning white and dying also. After the second week on incubation, the control shoot that rooted and had grown to about 4 inches tall. All of the plants looked healthy and the other 3 shoots has grown but had still not yet begun to form roots. In the T1 transgenic shoots, the one that looked to be sickly rooted and began to grow slowly to about 2 inches. The shoot that had already rooted was just as tall at 2 inches. Both plants look to be healthy and growing with green leaves. The other shoot looked to be dead, with brown shriveled tissue. The T2 transgenic shoots that rooted and growing, one faster than the other. One was about 1.5 inches tall while the other was 3 inches tall. The other 2 shoots died and had all shriveled white tissue. After the third week there was extensive growth in all rooted shoots with the control shoot hitting the top of the media container. The two T1 shoots are still growing more one now has a multitude of leaves while the other has one distinctly larger leaf. The T2 shoots are continuing to grow, one still larger than the other but growing in proportion to each other. As well the non-rooted shoots are all almost dead if not totally dead already. Part 5: Observation of gene expression The observation of gene expression was done by using the fluorescence microscope again except this time on some of the leaves from the plants grown up from the explants. Each plant at this point had rooted and grown a multitude of leaves or had died depending on its root status. The leaves for this part all came from the healthy growing plants in each container. The plants come up in red because of the chlorophyll in the leaves so we are looking to see if there is any green fluorescing showing us that the plant does contain and express the GFP gene that was inserted. In the control all of the leaf was pure red, no green showing at all. In the T1 transgenic plants, one (the plant with many leaves) had a very faint green tint to the plant but was mostly overpowered by the red. The other (with the one large leaf) had distinctly green veins in the leaf. In the T2 plants the taller plant also showed green veins throughout the leaf. The shorter leaf had darker veins but they were not exactly green, more just darker than the ruby red of the rest of the tissue. Part 3: Kanamycin assay The kanamycin assay gives us a lot of information on the nature of the seeds that the transgenic plant produces. These seeds were taken from a previous strain of Tobacco plants that under-went the same process as the tobacco plants in this experiment; the particular strain that I used was strain 1. After incubation for one week the germination rate was found to determine how well the seeds will
germinate on the selection media. The germination rate is found by dividing the number of seeds that germinated by the total number of seed planted. The formula is shown below here: Germination rate = Number of seeds germinated x 100% 34 x 100% = 68% Total number of seeds planted 50 Out of the 50 seeds that were planted on the selection media, only 34 seeds germinated giving the germination rate of 68%. Next we need to investigate the seeds that did germinate and look at the ratio of green shoots to white shoots. This ratio shows of the seeds that did germinate which ones contain the kanamycin resistance and which ones did not. The green shoots are the shoots with resistance and the white shoots do not contain the resistance gene. They turn white because they are dying. Of the 34 germinated seeds, 27 shoots were green and 7 were white; this gives us a ratio of 27:7. The next piece of information is the level of GFP expression among the germinated seeds with green shoots. The results of the GFP expression is shown below in table 1. Table 1: GFP fluorescence expression results Level of GFP Expression Number of seeds Low: 10% - 30% of area 12 Medium: 40% - 50% of area 8 High: 60% - 100% of area 7 The GFP fluorescence expression is a big part in determining how well the gene of interest is being expressed in the plant chromosome and also how many copies have been successfully inserted. The low level of expression was classified as have 10-30% of the area of the shoot that fluoresces green. The medium level is between 40-50% of area of the shoot that fluoresces, and the high level is above 60% of the area fluorescing green. The fluorescence was found using a blue light fluorescing microscope which showed the shoots as red where the green would then show up more easily to gauge. Discussion The results in part one and two are the expected results. The infection and co-cultivation went as anticipated and the result was calli forming on the explant that contained the transgene. The explant itself was supposed to die hence the brown and white coloration because the explant itself does not become transformed. Instead the calli that form, then produce shoots that are transgenic and will survive on the selection plate. Thus as the weeks went on the explant slowly die but the shoots that were produced by the calli formed dark green leaves and thrived on the selection media. It is also to note that some of those shoots are escapes which are seen later in part 4. The calli formed mostly at the mid vein because of the way that the plant transports material through the leaf. The bacterium would travel to the cut and form calli there faster because that is the most direct path to the cut. It is a natural road way inside the leaf. That is the explanation for why the calli at the midvein was so much bigger on each explant, and why some of the shoots produced at the midvein are escapes. Escapes happen because the plant forms a callus quickly and the shoot grows up and away before the selection media has a chance to act on the shoot. In this way the shoot can avoid selection and grow despite not being transgenic. The escapes were seen in part 4 with the rooting of the shoots because the
shoots were placed directly in the selection media to grow roots but instead they died because they were not actually resistant to the antibiotic in the media. After all of the shoots were rooted and had passed the selection test again we can see how many of the produced shoots were actually transgenic from each set. In the first set 2 out of 3 were transgenic and so from the total number of shoots we could make an educated assumption that 2/3 of the 240 total shoots would have turned out to produce transgenic plants, accounting for 160 total possible transgenic plants. In the second set 2 out of 4 successfully rooted and so 50% of the total 400 shoots could potentially make then 200 possible transgenic plants. Another important note is how well the plants rooted. Only the shoots in the control that did not root continued to grow, so we can see that the rooting rate in tobacco plants is actually only about 25% which is very low. It is odd then that all of the shoots that were transgenic in the transgenic sets did root. It could be due to bad selection of shoots from the control. There was a large amount of overgrowth on the control and so it was difficult to pick out specific shoots thus it could be due to human error in picking proper viable shoots and not just leaves. This same Fluorescent microscopy was done on the rooted plants that we had in part 4. In part 5 we also looked at gene expression not in the same way as the kanamycin assay but similarly. In part 5 we saw whether or not there was expression in the leaves of the transgenic and control plants that we transformed. The control showed no green fluorescence which is expected because we did not transform the control we wanted it to stay as a normal tobacco plant that was just regenerated in the same process of the transgenic plants. In the transgenic we see mostly green showing up in the veins of o the leaf. This is also expected because of the high amount of chlorophyll in the actual leaf tissue, so it would overpower the faint glow of the GFP in the microscope. However there is less chlorophyll in the circulatory system of the plant so the GFP can show more brightly thee because it is in every cell. Thus we can see that the plants are expressing the GFP gene. In some leaves it was not so clear but that is most likely because of either a lower expression of GFP or because of the chlorophyll once again overpowering the glow of the GFP. The GFP may have a lower expression because of an insertion before a repressor which would then repress the signal to produce GFP. Given the fact that the leaf that showed this came from the plant that had trouble rooting to start, this is a good explanation as to why it had trouble rooting and then showing expression. Overall it lived but it was slow to root and survive in the selection media because eth repressor not only repressed the GFP gene but also the kanamycin resistance gene. The kanamycin assay was the only part that did not deal directly with the rest of the experiment in terms of working with the same explants. The kanamycin assay was done using the previous year s tobacco plants that went through the same process. This does give us insight into what you would expect to see from our own transgenic plants in terms of self-pollination and seed germination. The assay was to see how well the seeds germinated and then whether or not the germinated seeds were expressing the gene of interest and also on what level they were expressing. First the seed germination rate was found to be 68%. This is a reasonable number given that some seeds are just not viable and perfect seed germination is rare among normal plants so in this case the germination rate is within an expected range.
The next piece of data is the ratio of green to white shoots after germination. The ratio was found to be 27:7. This ratio deals with the ratio of insertions within the plant genome. Because the plant is crossed with itself the ratio of offspring with the dominant trait is directly relatable to the number of insertions within the chromosome. With one insertion you expect a 3:1 ratio of green to white. With 2 inserts you expect a ratio of 15:1. This ratio is 27:7 which is very close to a 4:1 ratio. This ratio is much closer to a 3:1 than to a 15:1 ratio so here we can say that most likely the gene was only a single insert. This is to be expected because of the way the insert works. It does not insert into a specific spot so the more inserts you get the more likely that the gene will insert into a gene that codes for a trait that is critical to survival of the plant and thus kills the plant. So having the lowest number of insertions will give the best chance at germination and survival of the plant, thus you see more plants germinating with only a single insert. The last piece of information from the kanamycin assay is the level of expression for the shoots that were green from the germination. The majority of the shoots had low level expression, and the highest level of expression had the lowest amount of shoots. This is too expected given the results from the insertion ratio. The ratio was slightly higher than just the one insertion so it is more likely that some of the shoots had more than one insertion and thus the gene of interest was expressed twice giving it a much higher level of expression. However the ratio is not that much higher so the low level expression is most likely because of the way in which the fluorescence microscope works. Chlorophyll shows up red and the main part of the shoots was he leaves at the top which gives a bright red color due to the presence of chlorophyll in the leaves. The GFP would only really show up in the meristem and the new roots so that is not much area for the plant. The medium expression could be due to the insertion of the gene just before a promoter which would increase the amount of the GFP in the plant but not as much as having an extra insert. All-in-all the experiment went well as we were able to transform tobacco plants via agrobacterium and successfully regenerate a transgenic plant that expressed the genes that were inserted into its genome. Most of the experiment went as expected with some slight problems but nothing that was unexpected given the circumstances. References Bottino, Paul J., Dr. "Tobacco Leaf Disc Transformation Method." Tobacco Leaf Disc Transformation Method. The University of Maryland, 19 Feb. 2001. Web. 16 Apr. 2015. <http://www.life.umd.edu/cbmg/faculty/bottino/genetics/leafdisc.html>. Klee, H, Horsch, R, Rogers, S (1987) Agrobacterium-mediated plant transformation and its further applications to plant biology. Annu Rev Plant Physiol 38: pp. 467-486 McClean, Phillip. "Analyzing Plant Gene Expression with Transgenic Plants." Analyzing Plant Gene Expression with Transgenic Plants. North Dakota State University, 1998. Web. 16 Apr. 2015. <http://www.ndsu.edu/pubweb/~mcclean/plsc731/transgenic/transgenic2.htm>.