CLONING TREHALOSE SYNTHASE GENE AND ITS EXPRESSION IN Arabidopsis thaliana
Prapti Sedijani
BOGOR AGRICULTURAL UNIVERSITY 2007
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LETTER OF DECLARATION
Herewith I declare that the dissertation entitled “CLONING TREHALOSE SYNTHASE GENE AND ITS EXPRESSION IN Arabidopsis thaliana” is originally my work under supervision of the Supervision Committee. This manuscript has not been submitted to any University at any occasion. Any information mentioned in the text cited from published or unpublished of other parties are mentioned in the text and listed in the list of references.
Bogor, 11 June 2007
Prapti Sedijani P17600002
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ABSTRACT PRAPTI SEDIJANI. Trehalose is a well known as biological stabilizer functioning as stress protecting agent. It is ubiquitously found in bacteria, fungi, invertebrates, and certain desert-adapted resurrection plants such as Selaginella. In most flowering plants, trehalose is synthesized in amounts nearing the detection limit. Gene(s) encoding trehalose-metabolizing enzymes from E.coli, trehalose synthase and trehalose phosphate phosphatase (OtsA and or OtsB) and the same gene from yeast (TPS1 and or TPP) have been introduced to plants and increased stresses resistance, yet resulted in phenotypic defect, due to accumulation of its intermediate product, trehalose-6-phosphate (T6P). T6P increases along with the increase of trehalose level. This research was aimed to find out if introducing trehalose synthase gene (TreS) which does not produce T6P which prevent phenotypic defect resulted from trehalose accumulation, and whether the presence of TreS in plant improves plant resistance to stresses. The use of TreS as an alternative among non toxic selectable markers was also assessed. For this reason, genes encoding trehalose synthase originated from Thermobifida fusca (TfTreS) and originated from Mycobacterium tuberculosis (MtTreS) had been cloned into plant expression vector (pBIN1935S). TfTreS was isolated from T. fusca using PCR and was inserted to pGemT for sequencing. DNA sequent analysis of the 2 clones, TfTreS within pGemT were identical to the published data yet several silent point mutations occured. TfTreS from these clones were inserted into pBIN19 with CaMV35S promoter. It has been successfully transferred to Arabidopsis thaliana via Agrobacterium mediated transformation. MtTreS readily available in pBlunt TOPO II, was exiced from the plasmid and was inserted to pBin1935S, and transferred to Arabidopsis in the same manner, but it has not been successfully transferred to the plant. Selection of putative transgenic seedlings was carried out either on 50 mg/L kanamycin or on 125 mM trehalose containing media. Comparison of selection efficiencies using kanamycin or trehalose selection, it can be concluded that trehalose/TRES selection system allows for a tight selection yet yielded fewer (1/3 – 1/10) of transgenic lines than kanamycin selection system. Selection using a combination of trehalose and Validamycine A yielded transgenic lines yet at an even lower frequency (1/10) than trehalose selection. PCR analysis amplified TfTreS from all 5 transformed plants tested, and trehalose resistance was inheritable to the next generation. By growing transgenic plant on trehalose (100 mM) in combination with validamycin A (10 µM), it can be conclude that TfTreS can be used as an alternative of non-toxic selectable marker among the existing non-toxic selectable markers. Drought test was conducted as the initial test to see responses of transgenic plants to stresses. Thirty six percent of TfTreS expressing plants recovered 2 days after rehydration from 13 days withholding water. While from 7 days withholding water, 33% transgenic plants and 22% of wild type plants recovered. Enzymatic trehalose determinations using trehalase showed that some of transgenic plants contained high trehalose level ranged from 0-8.5 fold over the non-transgenic plants. Enzyme assay whitin phosphate buffer pH 6.5 consisted of 30% (833 mM) maltose at 25oC showed that TfTreS expressing plants had enzyme activity significantly higher than the Wt plants ranged from 025 folds. Cluster analysis based on leaf total water lost, leaf water retention, leaf dry weight, inflorescent trehalose level and inflorescent enzyme activity suggested that 33% of the transgenic plants withstand drought.
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ABSTRAK PRAPTI SEDIJANI. Trehalose dikenal sebagai agen stabilator terhadap materi biologi, berperan dalam perlindungan terhadap beberapa stres. Trehalose ditemukan pada bacteria, fungi, invertebrata serta tanaman yang telah beradaptasi dengan kondisi gurun, seperti Sellaginella. Kebanyakan tanaman berbunga,mensintesis trehalose dalam jumlah yang hampir tak terdeteksi. Gen penyandi enzyme metabolisme trehalose dari E. coli, trehalose phosphate synthase 1 and trehalose phosphate phosphatase (OtsA dan atau OtsB) and gen yang sama dari yeast (TPS1 dan atau TPP) telah diintroduksikan pada tanaman, namun dihasilkan kelainan fenotip, disebabkan oleh akumulasi trehalose 6 phosphate (T6P). Sebagai produk antara, T6P meningkat seiring dengan akumulasi trehalose. Penelitian ini bertujuan untuk mengetahui apakah introduksi gen trehalose synthase (TreS) yakni jalur yang tidak menghasilkan T6P sebagai produk antara, dapat mencegah timbulnya masalah yang disebabkan oleh akumulasi trehalose, dan apakah keberadaan TreS dapat meningkatkan ketahan terhadap beberapa stres. Kemungkinan penggunaan TreS sebagai salah satu alternative selectable marker juga di uji. Untuk itu, gen trehalose synthase dari Termobifida fusca (TfTreS) dan dari Mycobacterium tuberculosis (MtTreS) telah dikonstruksi dalam vektor expressi (pBIN1935S). TfTreS diisolasi denagn PCR. Analysis sikuen terhadap 2 klon TfTreS pada pGemT identik dengan data yang dipublikasi (), namun terjadi beberapa mutasi titik yang tidak bermakna. Gen tersebut telah dikonstruksi pada pBIN1935S, dan berhasil diintroduksikan pada Arabidopsis dengan bantuan Agrobacterium. MtTreS yang telah tersedia pada plasmid pCR Blunt II TOPO, dipotong dari plasmid tersebut, dikonstruksi pada pBIN1935S, dan ditransfer pada Arabidopsis dengan cara yang sama, namun belum berhasil terintegrasi pada tanaman tersebut. Seleksi tanaman transgenik putative dilakukan pada medium yang mengandung 50 mg/L kanamisin atau 125 mM trehalose. TRES/Trehalose merupakan system seleksi yang ketat dengan jumlah perolehan tanaman transgenik lebih rendah (1/3-1/10) dibanding dengan seleksi kanamisin. Sistem seleksi kombinasi trehalose dan validamisin A menghasilkan jumlah tanaman transgenik lebih rendah (1/10) dari kedua sistem seleksi tersebut. Analisis PCR menunjukkan bahwa TfTreS telah terintegrasi pada genom Arabidopsis pada semua (5) tanaman transformant yang di test. Resistensi tanaman transgenik terhadap trehalose bersifat menurun. Dengan tumbuhnya tanaman transgenik pada medium mengandung trehalose (100 mM) atau trehalose dan validamycin A (10 µM) menunjukkan bahwa TfTreS dapat digunakan sebagai alternatif selectable marker diantara non-toksik selectable marker yang telah ada. Test terhadap kekeringan dilakukan mengawali percobaan untuk melihat respon tanaman transgenik terhadap stres. Diperoleh 36% tanaman transgenik yang pulih setelah 13 hari tanpa irigasi. Sedang perlakuan seminggu tanpa irigasi, 30% tanaman transgenik pulih dari kelayuan berat dan 22% dari tanaman non-transgenik mampu pulih. Determinasi trehalose dengan pendekatan enzymatis menunjukkan bahwa terjadi akumulasi trehalose pada beberapa tanaman transgenik berkisar antara 0-8.5 kali rata-rata trehalose pada tanamanan kontrol. Aktivitas TRES tanaman transgenik dalam bufer fosfat pH 6.5 dengan substrate 30% (833 mM maltosa) menunjukkan bahwa aktivitas TRES pada tanaman transgenik secara significant lebih tinggi dalam kisaran 0-25 kali dibanding non transgenik. Analysis gerombol berdasarkan pada total air hilang pada daun, retensi air oleh daun, berat kering daun, kandungan trehalose dan aktivitas enzyme malai bunga menunjukkan bahwa adalah mungkin (33%) untuk mendapatkan tanaman transgenik yang bertahan pada kondisis kering.
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© Copy right belong to Bogor Agricultural University, the year of 2007 Copy right is protected
It is forbidden to copy and multiply partially or the whole of the dissertation in any form either reprint, photo copy, microfilm etc, without permission from Bogor Agricultural University (IPB)
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CLONING TREHALOSE SYNTHASE GENE AND ITS EXPRESSION IN Arabidopsis thaliana
Prapti Sedijani
DISSERTATION Prepared to fulfill the requirement to obtain a doctoral degree at the Department of Biology
BOGOR AGRICULTURAL UNIVERSITY 2007
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Title of dissertation
: CLONING TREHALOSE SYNTHASE GENE IN ITS EXPRESSION IN Arabidopsis thaliana
Name
: Prapti Sedijani
Registration number
: P17600002
Department
: Biology
Approved by Supervision Committee
Prof Dr. Ir. Edi Guhardja, M.Sc Chairperson
Dr. Dwi Andreas Santosa Member
Prof. Dr. Maggy T. Suhartono Member
Chairperson of The Study Program of Biology
Dean of Postgraduate School
Dr. Ir. Dedy Duryadi S., DEA
Date of Examination 19 June 2007
Prof. Dr. Ir. Khairil A. Notodiputro, MS
Date of passing Examination
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Acknowledgement AlhamduliLlah wa syukuriLlah, I would humbly express my deepest gratitude I addressed to Allah SWT for the life and all things supporting along with it, all the good will, the guidance and blessing that I always enjoy. The accomplishment of this dissertation is a part of His gives. He has chosen and allowed such many constructive and kind parties to contribute for the accomplishment of my study. Hence, via this opportunity, allow me to express my great gratitude and high appreciation to: Prof Dr. Edi Guhardja as The Chairperson of Supervision Committee for the guidance, knowledge, skills, all very kind sincere supports either material or immaterial all a long of my study. For the precious time, concerns and specifically for the outstanding wisdom that you always perform, I do thank you very much indeed. Dr. Dwi Andreas Santosa as Member of Supervision Committee for the guidance, knowledge, skills, all kind supports particularly the great laboratory supports with all the facilities that I have been used freely for such a long time. Without kind supporting laboratory, it would be nearly impossible to do research. Thank you very much indeed for the uncountable supports. Prof. Dr. Maggy T. Suhartono as Member of Supervision Committee for the guidance, knowledge, and skills that have been shared, I have been enjoyed the discussions that were very alive, criticisms, constructive, encouraging and the fruitful inputs that kept the work on the right trek. Dr. Henriette Schluepmann as Training Supervisor at Department of Molecular Plant Physiology (MPF), The Trehalose Project Leader Utrecht University, for the guidance, knowledge, skill and all kind supports in term of material and immaterial, the sincere concerns and very kind attention, during the laboratory works. I have been enjoyed the nice quality working together, that was very alive, criticism, constructive, encouraging and the fruitful. Thank you very much indeed. Dr Dedy Duryadi Solihin, the head of The Department of Biology, Postgraduate School, Bogor Agricultural University who allowed me to pursue PhD degree in the Department. Prof. Smeekens, the Department of MPF Utrecht University and as the Main Supervisor of the Training that endorse me to do research in the department and also provide me with all facilities during the training, thank you very much indeed. Dean of Postgraduate School of Bogor Agricultural University who allowed me to pursue PhD degree in the School. Prof. Dr. Mulyanto as The Former Rector of Mataram University and Ir. H. Mansur
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Ma’sum, MSc. PhD the current Rector of Mataram University who allowed me to be free from my duties in the university as well as for all kind supports so far for my study either material or immaterial supports. DUE-like Program of Mataram University, which provide scholarship enable me to conduct study at Bogor Agricultural University. Laboratory of Indonesian Centre for Biodiversity and Biotechnology (ICBB), The Chairperson (Dr. Dwi Andreas Santosa), The Head of Technical Laboratory (Mbak Sulastri) and all Staff members of laboratory that allowed me to do research conveniently with such nice and warm circular friendship. Laboratory of Molecular Plant Physiology Utrecht University and all the staffs that have been so helpful during my training in the department, thank you very much. Laboratory of BBBiogen, Chimanggu, and staffs that have been so helpful in trial of growing Arabidopsis, I do appreciate all the helps. Dr Yayuk Andayani, Dr Fauziah Harahap, Heru Kusdiyanto MSi/family, Zessi Menursita MSi, you all have been very supportive in term of material and immaterial all along of my study. Thank you very much for being nice and giving a warm circular friendship. Great appreciation also I addressed to friends and all parties that cannot be mentioned one by one, thank you very much for all the kind supports and warm circular friendship. Ibunda Siti Asiyah (the late) and Ayahanda Ismail (the late) there is no word can ever describes, no action can ever represents for all the scarifying, all the life, love and care, all sincere concern, all tears in all prayers that always been giving not only during my study but all along of my life. I always fill your presence in every moment of my time that keeps my spirit to fruit my life. All my brothers/sister (Mas Isnen M/the family, Mas Wiachto TM/the family, Mbak Triwahyuni/the family, Mas Terbit S/the family and Mas Sapto/the family) thank you very much indeed for your uncountable supports, sincere concern love and care, long life prayers for me that make my life easier, nicer, and better. With no means to discount the contribution of all parties, due to the limitation I own, this dissertation still lots of rooms for improvement. I invite the genuine constructive suggestions to make the dissertation better and useful.
Bogor, June 2007 The Author
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Curriculum Vitae
The Author was born in Magelang District at 11 July 1962 from a family of Ismail (my father) the late and Siti Asiyah (my mother) the late as the last born of a big family (6). Undergraduate school was completed from Institute of Teacher Training and Education of Yogyakarta (IKIP) in the year of 1986. In 1987 the author was formally accepted as an academic staff in The Department of Mathematics and Natural Science (Biology), Faculty of Teacher Training and Education, Mataram University. In the year of 1990, the author jointed a one year training program called Basic Science Bridging Program (BSBP) at Institute Technology of Bandung (ITB). Masters degree (M.Sc Hons) was obtained from Western Sydney University, Australia in 1997. The opportunity to pursue doctoral degree at Bogor Agricultural University (IPB) was started at the year of 2000. During the period of study at Bogor Agricultural University, she had an opportunity to undertake a one-year training course at Molecular Plant Physiology, Utrecht University, the Netherlands.
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LIST OF CONTENT
Title
Page
LIST OF TABLE .............................................................................................
xiii
LIST OF FIGURE ............................................................................................
xv
LIST OF APPENDIX ....................................................................................... xviii
I. INTRODUCTION 1.1 Rationale ......................................................................................
1
1.2 Objectives of the research .........................................................
2
1.3 Research Benefit .........................................................................
3
1.4 Hypothesis ...................................................................................
3
II. LITERATURE CITATION 2.1 Damaging effects of stress ........................................................
4
2.2 The character of trehalose and its role in several organisms
6
2.3 Trehalose, its role as stress protecting agent ..........................................................................
6
2.4 Other compatible solutes ...........................................................
11
2.5 Trehalose biosynthesis ..............................................................
12
2.6 Characterization of trehalose synthase (TRES/TSASE) on plant ..............................................................
16
2.7 Foreign trehalose synthase gene on plants ............................
17
2.8 Trehalose induce starch biosynthesis in Arabidopsis thaliana ...................................................................
18
2.9 Flow chart of the research .........................................................
21
III. CLONING GENES ENCODING TREHALOSE SYNTHASES from Thermobifida fusca and Mycobacterium tuberculosis 3.1 Introduction .................................................................................
23
3.2 Materials and methods ...............................................................
23
3.2.1 Place and time of research ........................................................ 3.2.2 Material of research ................................................................... 3.2.3 Cloning of TreS genes from Thermobifida fusca and from Mycobacterium tuberculosis ...................................................... Growth condition of Thermobifida fusca and DNA isolation ..... Cloning strategy .........................................................................
23 23 24 24 24
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Verifying TreS from Mycobacterium .......................................... Insertion of TreS gene to Plant Expression Vector ...................
25 25
3.3 Result and discussion 3.3.1 Cloning a gene encoding Trehalose synthase (TreS) from Thrmobifida fusca ............................................................. 3.3.2 Cloning a gene encoding Trehalose synthase from Mycobacterium tuberculosis (MtTreS) ......................................
33
3.4 Conclusion ...............................................................................................
36
26
IV. INTRODUCING TREHALOSE SYNTHASE GENE INTO Arabidopsis thaliana 4.1 Introduction .................................................................................
37
4.2 Material and methods .................................................................
37
4.2.1 Place and Time of Research ..................................................... 4.2.2 Material of Research ..................................................................
37 37
4.2.3 Preparation Agrobacterium solution for floral dipping ...............
38
4.2.4 Floral Dipping .............................................................................
38
4.2.5 Selection of transgenic lines ......................................................
38
4.2.6 Inheritability of TreS Expressing Lines ......................................
39
4.2.7 PCR analysis of TreS lines ........................................................
39
4.3 Result and discussion ................................................................
39
4.3.1 Frequency of putative mutants .................................................. 4.3.2 The presence of TfTreS within putative mutants ..................... 4.3.3 Inheritability of TreS Expressing Lines ......................................
39 42 43
3.4 Conclusion ...................................................................................
43
V. PRELIMUNARY TEST OF THE USE OF TRES AS A SELECTABLE MARKER 5.1 Introduction .................................................................................
45
5.2 Material and methods .................................................................
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5.2.1 Place and time of research ........................................................
46
5.2.2 Material of research ...................................................................
46
5.2.3 Assessment of TreS plants on trehalose or in combination with valydamycin A ........................................
46
5.3 Result and Discussion ...............................................................
47
5.4 Conclusion ..................................................................................
53
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VI. CHARACTERIZATION OF TRES EXPRESSING LINES 6.1 Introduction .................................................................................
55
6.2. Material and method ..................................................................
55
6.2.1 Place and time of research ........................................................ 6.2.2 Material of research ................................................................... 6.2.3 Characterization of TreS line ..................................................... Growing Arabidopsis thaliana .................................................... Enzymatic trehalose determination ........................................... Determination of enzyme activity ............................................... TfTreS expressing lines in response to drought ........................ Leaf water retention and leaf recovery test .............................. In planta TfTreS expressing plants in response to drought .......
55 55 55 55 56 56 57 57 57
6.3 Result and discussion 6.3.1 Growing Arabidopsis thaliana in tropical climate ....................... 6.3.2 Trehalose content and enzyme activity of TfTreS expressing lines ......................................................... 6.3.3 Enzyme of TfTreS Expressing lines ........................................ 6.3.4 Trestf expressing lines in response to stress ............................ Leaf Water Retention .............................................................. Cluster Analysis based on parameters observed ..................... Leaf Recovery After 16h RT incubation ................................ TfTreS expressing lines in response to drought .......................
57 60 60 62 62 65 69 71
6.4 Conclusion ...............................................................................................
76
VI. GENERAL DISCUSSION ..........................................................................
77
IX. CONCLUSION AND RECOMMEDNDATION ..........................................
81
XI. LIST OF REFERENCE ..............................................................................
82
XII. APPENDIXES ...........................................................................................
92
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LIST OF TABLE
Title
Page
Table 1. Frequency of putative mutants selected on kanamycin or trehalose
41
Table 2. Frequency of trehalosa resistant seedlings of T1 dan T2 ................
43
Table 3. Frequency of resistant seedling to 125 mM trehalose and 10 mM validamycin A .................................................................
47
Table 4. Number of T3 seedlings resistant to 100 mM trehalose ..................
60
Table 5. Trehalose content of published some transgenic plants ..................
62
Table 6. Leaf water lost at interval of time after detachment .........................
63
Table 7. Detached Leaf Total Water Loss and Leaf Dry Weight ....................
65
Table 8. Observed characters on leaves after detachment ...........................
66
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LIST OF FIGURE Title
Page
Figure 1. The damaging effect of heat to membrane and the role of trehalose to decrease the effect ....................................
10
Figure 2. Trehalose biosynthesis ....................................................................
14
Figure 3. Trehalose pathways in organisms ...................................................
15
Figure 4. Starch breakdown in chloroplast and carbon export at night ..........
19
Figure 5 PCR Product at various annealing temperature ..............................
27
Figure 6. Schematic Diagram of pGemT reS ..................................................
28
Figure 7. Restriction analysis of plasmid isolated from E. coli colonies expected to bear TreS gene ...................................
29
Figure 8. Restriction analysis based on computer simulation of TreS sequence .................................................................................
30
Figure 9: Sequence of TreS from Thermobifida fusca cloned in E.coli ..........
31
Figure 10. Schematic diagram of pBIN1935STreS ........................................
32
Figure 11. Restriction analyses of TreS inserted in the plant expression cassette of pBin1935S .....................................................................
33
Figure 12. Restriction analysis of TreSMtbc within pCR Blunt II TOPO (A)
34
Figure 13: Schematic diagram of pBin1935SMtbc .........................................
35
Figure 14. One-week-old putative TfTreS transformed seedlings selected on selection medium .........................................................
40
Figure 15. Seedling resistant to kanamycin or trehalose ...............................
42
Figure 16. PCR product of putative mutant ....................................................
42
Figure 17. Selection of putative mutants on trehalose/validamycin A ...........
42
Figure 18. Seedling growth on validamycin A ................................................
47
Figure 19. TfTreS lines plants grown on various medium ..............................
51
Figure 20. Rice callus grown on sugars .........................................................
52
Figure 21. Grown Arabidopsis in tropical area ...............................................
58
Figure 22. T3 plants selected on trehalose ....................................................
59
Figure 23. Trehalose concentration and enzyme activity of TfTreS expressing lines and its control plants ..........................
61
Figure 24. The average of excised leaf water loss at pointed time ................
63
Figure 25. Drought tolerance test using leaf water retention measurement method ...................................................................
64
Figure 26. Cluster diagram of detached leaves based drought parameters...
66
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Figure 27. Leaf recovery test of dehydrated leaves .......................................
67
Figure 28. The trehalose content / enzyme activity of recovered leaves from drought .....................................................
68
Figure 29. TfTreS expressing lines against drought .....................................
72
Figure 30. TfTreS expressing lines against drought (2nd experiment) ...........
73
Figure 31 Plants recover from drought ..........................................................
74
Figure 32. Fresh and dry weight of plant recovered from drought .................
75
Figure 33. Starch staining of seedlings grown on trehalose or ½ MS ...........
79
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LIST OF APPENDIXES
Appendix 1. TreS from several organisms ....................................................
92
Appendix 2. Codon usage of AtTPS1 of Arabidopsis thaliana and TreS of Thermobifida fusca .......................................................
93
Appendix 3. Trehalose content and enzyme activity of TfTreS expressing plants and Wt plants ..................................
93
Appendix 4. Regression analysis of standard curve: glucose versus optical density and graphic plot ........................
94
Appendix 5. T-Test of Trehalose Content or Enzyme Activity between Wt and TfTreS plants .................................................
95
Appendix 6. Box lot analysis of Trehalose content or Enzyme activity between Wt and TfTreS plants ................................................................ 96 Appendix 7. Site mutation of TfTreS found in the gene after being cloned in E. coli .............................................................
97
Appendix 8: Single factor analysis of water loss at pointed time ..................
98
Appendix 9. Leaf Total water loss .................................................................. 100 Appendix 10. Single factor ANOVA of leaf Dry Weight ................................. 100 Appendix 11. Fresh and Dry Weight of Tested plants
................................ 101
Appendix 13. Single Factor Anova of Frequency of Putative Mutants Selected on Kanamycin or Trehalos .......................................... 102 Appendix 14. Letter of permission ................................................................. 103
