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AHMAD FIQRI MUSTAQIM OTHMAN 4/14/2014

982440046
LAB REPORT ON TOBACCO TRANSFORMATION
INTRODUCTION
Tobacco is one of the plant species from genus Nicotiana of the Solanaceae family. There are
many usages of tobacco in our society such as cigars, cigarettes and others
1
. However, tobacco also
commonly used in plant transformation as it is easy to culture, cheap and has a fast regeneration
process. These traits make tobacco plant one of the good plant model system for plant transformation.
Since we are going for a natural plant transformation system, we used the bacterium Agrobaterium
tumefaciens. Agrobacterium tumefaciens; (tumefaciens means tumor making) is a good model for
bacterium in plant transformation as it easy to modify its plasmid and cheap. In order to ensure that the
offspring from the original plants were fully transformed, they would have to undergo screening and
selection. The selector gene that we used in this experiment was Kanamycin and Moxalact gene. As for
the reporter gene, we used the GFP and GUS genes. Our objective for this experiment is to study the
tobacco transformation via agrobacterium-mediated tobacco transformation as well as its shoots and
roots formation.



METHOD AND MATERIALS

1
"Tobacco." Wikipedia. Wikimedia Foundation, 15 Apr. 2014. Web. 16 Apr. 2014.
<http://en.wikipedia.org/wiki/Tobacco>.


Agrobacterium-mediated tobacco tumefaciens uses the soil bacterium Agrobacterium
tumefaciens. Agrobacterium tumefaciens can infect most dicotyledon plants via wounded sites.This
allow the Ti (tumor-inducing) plasmid that has been genetically modified in the bacterium transmitted
into the plant tissues
2
. The incorporated Ti plasmid can then integrate its gene into the chromosome of
the plant which will cause the regenerated tissues around wounded area to develop a neoplastic growth
called crown gall tumor
3
. In this experiment, we used a media has kanamycin and moxalacatam for
selection process. Kanamycin is used to select for transformed plants while Moxalactam is for killing any
Agrobacterium that present in the explants. After we have the transformed explants, we test for its
regeneration of shoots and roots. In shoot regeneration, selection was done with a media containing
Kanamycin while using the seeds from self-pollinated transgenic plants (RO). For the regeneration of
shoots and roots, we calculate its regeneration efficiency by using GFP observation and germination
segregation. Form these, we can know how efficient Agrobacterium tumefaciens in transforming
tobacco plant.




RESULTS
Part 1-Initiation transformation observation:

2
"Plant Genetic Engineering: Methodology." Plant Genetic Engineering: Methodology. Center for Bioenergy &
Photosynthesis, 16o Oct. 2006. Web. 16 Apr. 2014.
<http://bioenergy.asu.edu/photosyn/courses/BIO_343/lecture/geneng.html>.
3
Razdan, M. K. "13.3.3/genetic Transformation." Introduction to Plant Tissue Culture. 2nd ed. Enfield, NH: Science,
2003. 180. Print.



At this point there were not much of results can be seen, only observation on the leaves site of
injuries. There were not much of a difference between control and transgenic explants at this
time, most of them are pretty much the same at this point.
- There are some cuts on the veins of the leaf and a gel-like substances coming connecting the
cut.
- Tricons were seen at the exterior of the leafs
- The color is paler at the site of the wounds
- There are bubbles at the site of injuries
Conclusion: The observation is consistent to the standard operation when a plant is wounded.
Part2-Selection and screening
Control:
o there were no callus formation yet
o Most of the cuts were closed
o Some of them has an increase in size and the others do not change in size.
o There are 86 shoots from the three explants
- Transgenic:
o There were some callus formation on the explants
o There are not much difference in term of size
o The color looks paler than previous
o (a) contains 82 shoots while (b) contains 95 shoots from the three explants each
GFP expression screening
- Scale for GFP concentration:


0= no GFP, 1= very low spread of GFP, 2= medium spread of GFP, 3= high spread of GFP
GFP concentration
explants Control Transgenic (a) Transgenic (b)
1 0 0 1
2 0 0 0
3 0 0 0
4 0 0 0
5 0 0 0

Shoot organogenesis efficiency ratio
control Transgenic (a) Transgenic (b)
Total explants 3 3 3
Total shoots 86 82 95
Efficiency ratio 28.7 27.3 31.7

Conclusion: Callus formation in the transgenic explants suggest that the new transformed plants
are being produced.


Part 4- Rooting of tobacco shoots


Part 4 have to be done first as our explants growth was too fast and in order to prevent wilting
from malnutrition. The results for part 5 have been compiled here to allow better understanding
in root organogenesis process. These are the results for the experiment:
- Most of the shoots are growing well and the color looks good
- There were a couple of leaves that lost contact with the media but some still have root
formation.
Root organogenesis efficiency ratio
control Transgenic (a) Transgenic (b)
Total explants 5 5 5
Total roots 36 23 18
Efficiency ratio 7.2 4.6 3.6

Conclusion: Even though the growth of these shoots is good, we have a low rooting efficiency.
Part 3- Seed germination assay
- Only 12 seeds out of 62 seed doesnt germinate at all
- There were 30 seeds that have germinated have green color and big
- There were 11 seeds that have germinated have white color and small
- Kanamycin segregation ratio
= # of green color seeds that germinated/ # of white color that germinated
= 30/11
= 2.7
o This is consistent with the 3:1 segregation ratio


- GFP segregation ratio
= (# of germinated seeds that express GFP)/ (# of germinated seeds that do not
express GFP)
= 26/4
= 6.5
o This is closer to 15:1 segregation ratio
o Conclusion: In the Kanamycin segregation ratio, the result suggest that we have one
insertion while based from the GFP expression segregation ratio, it suggest we have
two insertions.
DISCUSSION
Based from these results, I would say that the overall result is quite good as most of them are
similar to the standard. For part 1, we can see that there were no callus formation on the control while
there were quite a few on the transgenic explants. This is simply because of the Agrebacterium
tumefciens Ti plasmids that got inserted into the transgenic plants via the wounded site. As explained
above, the Ti (tumor inducing) will get incorporated into the plant chromosome and lead to formation of
callus.
Since the tobacco explants were put under the selection for Kanamycin and moxalactam, we
expect to have more transgenic plants. We can determine he ingenuity of the transgenic plants by
checking its GFP expression. GFP was one of the selectable marker for our transformation experiment,
this means that only a transgenic plants will express GFP. However, based from the screening, I can say
that not all of the transgenic explants were truly transgenic at all since only one of the transgenic (b)
explants have GFP expression. This could happen when some of the explants manage to escape the


selection of Kanamycin and Moxalactam. However, we did get a decent amount of shoots formed in this
part 2 experiment.
In part 3, the segregation ratio between kanamycin and GFP expression is different can be
explain by a number of reasons. First, it could be due to gene silencing. This could happen during
transcriptional or post transcriptional stage, due to promoter methylation or methylation of the coding
sequence respectively
4
. Secondly, the difference in segregation ratio could be due to random location
during the insertion for example, the closer the insertion to the promoter or enhancer, the more the
expression of GFP a plant has and vice versa. Thirdly, it is possible that different individuals have
different fertilization process. These are all possible results as why the Kanamycin segregation ratio is
different than the GFP segregation ratio.
As for part 4 and 5, the rooting efficiency seems low could probably due to some of the explants
were not in contact with the media. This probably happens from an improper handling of the magenta
boxes causing the explants to be detached from the media. However, from those explants that were
actually attached to the media, their roots growth is amazing. This could probably due to the selection
of explants for rooting, the best leaves were chosen and the vitrified leaves were avoided. This
promotes the growth of the best line of offspring.



CONCLUSION

4
"Gene Silencing in Plants." What Is The Biotechnology. N.p., 21 Oct. 2013. Web. 16 Apr. 2014.
<http://www.whatisthebiotechnology.com/blog/gene-silencing-in-plants/>.



In the academia level, Agrobacterium-mediated tobacco transformation is probably the best
plant transformation system there is. It is cheap, easy and does not take too much time. Furthermore,
the results may not have been embracing due to human error but the protocol used for this experiment
was proven to be successfully transforming the tobacco plant and having a complete shoot and root
organogenesis.
The results for this experiment can be improve by ensuring that the students truly understand
the methods and ways to improve sanitation during the experiment. This includes reducing the amount
of talking when doing lab works that involve the laminar flow hood, always sanitize the workplace and
always remember to organize ones work place before starting the experiment. Next, the explants in this
experiment were placed in a room under the basement in South Frear. The room has been said does not
have an accurate and stable condition in terms of lighting, humidity and temperature as there is a
construction going on in the basement. Fluctuation in these area could have affect the growth of the
plants which will in turn affect the result of the experiment.







BIBLIOGRAPHY


1. "Tobacco." Wikipedia. Wikimedia Foundation, 15 Apr. 2014. Web. 16 Apr. 2014.
<http://en.wikipedia.org/wiki/Tobacco>.
2. "Plant Genetic Engineering: Methodology." Plant Genetic Engineering: Methodology. Center for
Bioenergy & Photosynthesis, 16o Oct. 2006. Web. 16 Apr. 2014.
<http://bioenergy.asu.edu/photosyn/courses/BIO_343/lecture/geneng.html>.
3. Razdan, M. K. "13.3.3/genetic Transformation." Introduction to Plant Tissue Culture. 2nd ed.
Enfield, NH: Science, 2003. 180. Print.
4. "Gene Silencing in Plants." What Is The Biotechnology. N.p., 21 Oct. 2013. Web. 16 Apr. 2014.
<http://www.whatisthebiotechnology.com/blog/gene-silencing-in-plants/>.