were introduced into Tobacco and sugar cane also make the transgenic plants tolerated to drought, salt and cold Zhang et al 2005; Zhang et al 2006.
To be able to use TfTreS as a selectable marker, the transgenic plants have to have a very strong activity to combat the effect of high level trehalose on
the medium. This experiment tested the capability of TfTreS expressing lines to grow on medium supplemented with trehalose and trehalase inhibitor
Validamycin A. Validamycin A is an antibiotic that inhibits trehalase activity, therefore, trehalose added to the medium is remained intact, and endogenous
trehalose level within the tissue can be increased Godijn et al. 1997, Muller et al. 2001. When the plants resistant to trehalose or trehalose and validamycin A in
such combination that the Wild type plants are unable to survive, it means that TfTreS
can be used as a selectable marker on trehalose or trehalose and validamycin A.
5.2 Material and methods 5.2.1 Place and time of research
This research was conducted in Laboratory of Molecular Plant Physiology MPF, Utrecht University, The Netherland Mei-July 2005.
5.2.2. Material of research
Seeds of Arabidopsis harvested from T0 plants, namely plants that have been subjected to floral dipping previous work Chapter 4.
5.2.3 Assessment of TfTreSplants on trehalose and validamycin A.
About 40 mg seeds 2000 seeds harvested from transformed plants T1 were grown in ½ MS supplemented with trehalose and Validamycin A. To obtain
the optimal combination of both trehalose and Validamycin A, kill curve of wild type seeds was done prior to the actual treatment. In kill curve experiment,
combination of 10, 50 and 100 µ
M validamycin A and trehalose at 50 and 100 mM trehalose were used. As control, seeds were also grown on ½ MS medium.
Observation was made at 1 and 2 weeks after exposure to light. To obtain the very strong prove of TfTreS as a selectable marker, we did a trial to used 125
mM trehalose and 10 µM vlidamycin A combination applied to T1 seeds. When T2 was available, the seeds were subjected to 100 mM trehalose and 10 µM
validamycin A.
5.3 Result and Discussion
Kill curve of Wt seeds on combination of 10, 50 or 100 µ
M validamycin A and 50 or 100 mM trehalose in the medium was done. It was observed that 1-
week-old Wt seedling develop shoots and roots as normal growth, while those grown on all combination of trehalose and validamycin A showed similar severe
growth. After 2 weeks the seedlings on any combination were died Figure 17A. This phenomenon suggested that either 50 or 100 mM trehalose in combination
with 10 µM validamycin A should be sufficient to kill the wt seedlings. This suggested that the lowest combination 10 µM validamycin A and 50 mM
trehalose is enough to select the transgenic plants bearing TfTreS. To make sure,
Figure 17: Selection of putative transgenic TreS using trehalose and validamycin A. A trial using Wt seedlings grown on combination of Trehalose and
validamycin A. All combination of 10, 50 and 10 µM validamycin with 50 and 100 mM trehalose kill seedlings, while the control remained fresh
A. T1 seedling grown on 125 mM trehalose + 10 µM validamycin A B, root site view of transgenic plants is presented, with long roots
shown by arrows C. T= trehalose, and V= validamycin A. 100T and 100 V containing plate is not shown.
Table 3. Frequency of T1 seedling on 125 mM trehalose 10 µM Validamycin A for every 40 mg seeds 2000 seeds
Plate 816 918 wt 920
1 2
3 4
5 6
7 8
9
10 1
2 2
1 1
2 1
1 2
2 1
2
Average 1
1
A
C A
B
0 T 0 V
50 T 10 V
50 T 50 V
50 T 100 V
100 T 10 V
100 T 50 V
however, we tried to put the T1 seedlings because there has been available T2 on combination of 10 validamycin A and 125 mM trehalose. The result is
presented in Figure 17 B-C. The frequency of seedling resistant to this combination is very low Table 3. It is questionable whether validamycin A
contributed to the selection performance. To clarify the question, about 40 mg seeds were grown on ½ MS supplemented with 50, 100 or 500 µM validamycin
A. Observation was made 1-2 weeks after light exposure. The result Figure 18 showed that seedlings were in vigorous growth at 12MS. Shoots growth were no
affected by validamycin A up to 100 µM, while roots growth were progressively reduced when 50 µM validamycin A is added. While those grown on 500 µM
Validamycin A showed severely affected. It germinated at 8-9 days incubation, followed by slow growing with shoots and root stunted. This suggested that
validamycin A it self render root growth. Kill curve revealed that a combination of 10 µM validamycin A and 50 mM trehalose is enough to kill all the seedlings. It
might suggest that the combination can be used as selection agent to select putative transgenic Arabidopsis seedling bearing TfTreS gene.
When T2 of TfTreS expressing line was available, some TfTreS expressing plants were subjected to combination of 100 mM instead of 125 mM trehalose in
combination with 10 µM validamycin A.
Figure 18. Seedling growth on validamycin A, at ½ MS medium A 50 µ
M B, 100
µ M validamycin A C, 500
µ M validamycin A D. Shoot growth
seemed to be similar, but roots were progressively reduced with the increase of validamycin A. At 500 µM, validamycin A, delayed
germination at 8-9 days days after sawing.
A
B C
D
The result is presented in Figure 19. Unfortunately, however, the frequency was not recorded, but the Figure clearly shows that almost all lines of clone 916
resistant to the combination, while cloned 816 that resistant to trehalose show less vigor growth than clone 918 on the combination. Trehalose at 100 mM it self
can be used to select TfTreS transgenic Arabidopsis Figure 19. Addition of Validamycin A in combination with trehalose increases the toxicity of trehalose to
plants. The TfTreS expressing lines, furthermore, the transgenic plants grow in quite wide range of trehalose with the presence of 10 µM validamycin A et least
it was observed at 125 mM trehalose and 10 µ
M Validamycine A. The strength of the TfTreS activity of interest might be adjusted by manipulating the
concentration of trehalose and validamycin A as the selection agents. Question rose whether it is applicable for other species. Casper and
Schluepmann 2006 personal communication revealed that rice might contain high level trehalase. It deduced from kill curve of non transgenic Japonica rice
callus that still can grow on 100 mM trehalose, although it is not as vigor as on glucose. When Validamycin A is added, the growth was severely inhibited.
Hence, it might be suggested that trehalose can be used as selection agent when endogenous trehalase is low. The use of trehalose in combination with low level
validamycin A increases the selectivity of trehalose. The convertion of trehalose into maltose is not inhibited by validamycin A, hence allow growth of engineered
materials e.g. seedlings or callus bearing TfTreS. Many markers genes available, but only three are used in more than 90 of all cases Miki and McHugh 2004.
They are kanamycin and hygromycin antibiotics and phosphinothrycin herbicide resistance genes by which their proteins alter antibiotics herbicide.
Environmental question about the use of these marker genes are the possibilities of gene flows from transgenic crops to the other croprelative crops and
horizontal transfer from transgenic crops to other organism. Non toxic selectable markers act as in such a way that are more natural to
the plant have also been developed such as targeting plant’s metabolism and altering plant’s development. These might be fit to the public concern about
antibiotic resistance genes that may increase human and animal tolerance to antibiotics; and herbicide resistance gene that may create environmental
problems. Some example of non toxic selectable marker are: the use of galactose transferase gene, D-amino acid oxidase gene and phosphor manose
isomerase manose gene. E. coli UDP-glucose: Galatose-1-phosphate
uridyltransferase galT gene converts galactose to glucose. Almost all plant including agronomic crop, such as potato, maize and wheat are sensitive to
galactose. The expression of the gene allows selection of the transgenic plants on galactose containing medium Osborn 2007, www.ippragmetics.com. D-
amino acid is the optical isomerase of L-amino acid. D-amino acid is metabolized via oxidative deamination catalyzed by D-amino acid oxidase DAO that is
encoded by DAO1 gene. The enzyme is missing in plant. Plant, metabolizes D- amino acid that lead to the production of N-malonyl and N-acetyl derivates. The
conjugation of both might result in a toxic compound. By selecting the amino acid used, it is allowed to do selection positively or negatively, since some D amino
acid are metabolized that lead to growth acceleration Lamlin et al. 2007 www.ncbi.nlm.nih.gov. While phosphomanose isomerase PMI is an E. coli
enzyme encoded by pmi gene. The enzyme catalyzed conversion of mannose 6 phosphate, an inhibitor of glycolysis into fructose phosphate that is glycolysis
intermediate product. The cell is selected on mannose containing medium. Frequency of mutant as much as 2.5 of treated seeds were obtained using
mannose isomerase applied on Arabidopsis thaliana with increased 5 fold ranging level of enzyme activity Todd and Togue 2001.
Marker free transgenic plants, however, might be ideal transgenic plant to answer public question about biosafety of transgenic plant, simply to shorten the
regulatory processes, and allows the use of markers that has not been extensively tested Anonym, www.inspection.gc.ca. Darbani et al. 2007
suggested that there are two ways of producing marker free transgenic plant, 1 by using non-toxic marker gene, and 2 by excising the marker. Our study tried to
use TfTreS as an alternative selectable marker. Trehalose synthase converts maltose into trehalose, yet it converts trehalose into maltose when it reaches
equilibrium state that is about 60 of trehalose. As discussed in the previous chapter that Wt plant could not survive on high level of trehalose such as due to
starch immobilization from source to the sink. TRES activity that allows reverse back trehalose into maltose give energy allowing transgenic plant survives on
high level trehalose. TfTreS expressing plants have higher capability to slow down water lost, withstand to drought, and have higher dray matter than the Wt
plant. As trehalose is widely used in food ingredient, cosmetic, and in medical treatment, it might be safe when TfTreS gene applied to crop plants.
Figure 20. Rice callus grown on sugars. Vigor growth of rice callus on glucose,
but slowly growth trehalose, and no growth when validamycin A was added to medium contained trehalose was observed A. The figure was
provided by Casper and Schluepmann 2006, personal communication. Fresh weight of callus growth on the corresponding media is presented
B. S= sucrose, T = trehalose, TV10 = Trehalose + 10 mM validamycin A, TV20= trehalose + 20 mM Validamycin A and G = glucose.
Furthermore, trehalose is degraded in human intestine, since trehalase, the enzyme that degrades trehalose into glucose is presence. Therefore, using
TfTreS as selectable marker might be considered as producing marker free
transgenic plant. The use of validamycin A included in the selection system does not harm to
plant, instead of helping plants against several plant diseases due to bacteria and fungi, e.g. Chinese cabbage soft rot, cabbage black rot, lettuce bacterial rot
Ishikawa et al. 2005. They further report that Validamycin A also control over diseases caused by Xantomonas champestris, Psudomonas cichori, Rizoctonia
solani and Fusarium. Foliar spraying using validamycin A induces accumulation
salicylic acid SA and controls tomato Fusarium wilt. This might also due to validamycin A inhibits trehalase activity, hence trehalose is accumulated in the
hyphae, and it’s growth is inhibited see Ishikawa et al. 2005. From the above mentioned, it might be concluded that TfTreS can be used
as an alternative non-antibiotic selectable marker on trehalose for those having low trehalase activity e.g. Arabidopsis or trehalose in combination with
validamycin A for those having a high trehalase activity e.g. rice callus. Using TfTreS
as a selectable marker may be beneficial, since its product is edible, environmentally and biologically friendly. The presence of the gene also indicated
that the transgenic plants might withstand stresses. This capability is also supported by other role of trehalose that recently proved to involve in enhancing
of transcriptional activity of heat shock protein gene Hsf1 and increase phosphorylation of the enzyme Collin and Nelson 2007. Trehalose also act
synergistically with late embryonic abundant o protein LEA which prevent protein aggregation in stressed condition Goyal et al. 2005. Tobacco and
sugarcane bearing TSase from Grifola frondosa Zhang et al. 2005; Zhang et al. 2006 produced normal growth and increased plant resistance to drought, salt
and cold. Hence, using the third pathway of trehalose synthesis genes might overcome morphological defect due to high trehalose level as well as increased
the resistance of transgenic plant on drought, salt and cold. The presence of TfTreS
in Arabidopsis also indicated the improvement of plants withstand drought Chapter 6.
5.4 Conclusion
The presence of TfTreS in Arabidopsis allows plants to resist high level trehalose that lead to the possibility of the use of TfTreS as a non-toxic selectable
marker. Using TfTreS as a non toxic selectable marker might be beneficial and
meet of the public concern about bio safety for it is human, environment and biologically friendly, as well as indicated the increase of the capability of
transgenic plants withstand stresses. The presence of validamycine A in combination with trehalose increases selectivity of the selection system to obtain
transgenic plant. Trehalose 100 mM is a tight selection agent for Arabidopsis thaliana
beraing the gene.
