Flower development of Lilium longiflorum - The Lilium information ...

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Chapter 5 The transformation experiment used about 900 explants divided over 15 dishes. The bombardment was carried out using the Biolistics PDS 1000/He system (Sanford et al., 1993) using 1.0 µm gold particles (Bio-Rad, Veenendaal, the Netherlands) as a carrier. Three milligrams of particles were coated with 5 µg of plasmid DNA (in a 60 µL solution) and 7.5 µL was used for each bombardment. Experiments were set up with 1 shooting at 1100 psi (treatment 1x1100) on about 400 explants or 3 shootings at 1800 psi (3x1800) on around 500 explants using a target distance of 9 cm. Additionally, 50 explants per treatment were bombarded with the control plasmid, pPG5. Selection and identification of transgenic plants. Plants were kept on propagation medium supplemented with 2 mg L -1 Basta ® to select for plants constitutively expressing the PAT gene. The regenerated plants were tested for the presence of the transgenes by PCR. Additionally to the Basta ® resistance, the presence of PAT was identified by a PCR product amplification using the oligonucleotides 5’-GAT TAG GCC AGC TAC AGC AGC-3’ and 5’-CCT TGG AGG AGC TGG CAA CTC-3’ as forward and reverse primers, respectively, at an annealing temperature of 65 o C. The presence of SUP in the genome was recognised by the amplification of a 613-bp fragment using the gene-specific primers 5’-ATG GAG AGA TCA AAC AGC-3’ (forward) and 5’-TTA AGC GAA ACC CAA ACG-3’ (reverse) using 55 o C for the annealing step. The genomic lily DNA was extracted using the method described by van Heusden et al. (2000) and 500 ng were used in the PCR reaction. Explants bombarded with pPG5 were assayed for transient β-glucuronidase activity after 5 days after bombardment using standard conditions (Jefferson et al., 1987). Transgenic plants were kept in vitro until their bulblets were 2-3 cm diameter to be vernalized and transferred to soil under greenhouse conditions. These plants were followed by 2 flowering seasons under standard environmental conditions for flowering production in the greenhouse. Assessing transgene expression. Floral meristems and leaves derived from transgenic lily plants growing in greenhouse conditions were harvested. They were immediately frozen in liquid nitrogen and kept in –80 o C for further analysis. Total RNA was extracted by the Zhou et al. (1999) method. Northern blot, probe synthesis and purification, and hybridisation procedures were as described elsewhere in this thesis. 76 Constitutive transcription, for RNA loading control in northern blots, was
Transformation of Lilium via particle bombardment assessed through the expression of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene, that participates in the glycolysis pathway. A 620-bp fragment of the lily GAPDH was amplified by RT-PCR using the degenerated primers (5'-GTK GAR TCN ACY GGY GTC TTC ACT-3' and 5'-GTR TGR AGT TGM CAN GAR ACA TC for forward and reverse annealing, respectively), cloned in pGEM-T-easy vector (Promega, Madison, WI), sequenced and used as a probe. Acknowledgements The authors would like to express their appreciation to Jurriaan Mes for his efforts during the early stages of this work and to Santie de Villiers for the valuable suggestions and discussions. This work was supported by CNPq, the Science and Technology Development Organ of the Brazilian Government (200851/98-5 GDE) and by the Dutch Ministry of Agriculture, Nature Management and Fisheries (DWK 364). REFERENCES Angenent GC, Franken J, Busscher M, Colombo L, van Tunen AJ (1993) Petal and stamen formation in petunia is regulated by the homeotic gene fbp1. Plant J 4: 101-112 Aswath C, Nhut DT, Bui VL (2001) Lilium. In: Parthasarathy VA, Bose TK, Das P (Ed.) Biotechnology of Horticultural Crops. Vol. 3, Naya Prokash, Calcutta, p.134-168 Bereterbide A, Hernould M, Castera S, Mouras A (2001) Inhibition of cell proliferation, cell expansion and differentiation by the Arabidopsis SUPERMAN gene in transgenic tobacco plants. Planta 214: 22-29 Bowman JL, Sakai H, Jack T, Weigel D, Mayer U, Meyerowitz EM (1992) SUPERMAN, a regulator of floral homeotic genes in Arabidopsis. Development 114: 599-615 Coen ES, Meyerowitz EM (1991) The war of the whorls: genetic interactions controlling flower development. Nature 353: 31-37 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS-fusions: glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901-3907 Kanno A, Saeki H, Kameya T, Saedler H, Theissen G (2003) Heterotopic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana). Plant Mol Biol 52: 831-841 Kater MM, Franken J, van Aelst A, Angenent GC (2000) Suppression of cell expansion by ectopic expression of the Arabidopsis SUPERMAN gene in transgenic petunia and tobacco. Plant J 23: 407-413 Langeveld SA, Gerrits MM, Derks AFLM, Boonekamp PM, Bol JF (1995) Transformation of lily by Agrobacterium. Euphytica 85: 97-100 Lipsky A, Cohen A, Gaba V, Kamo K, Gera A, Watad A, Hammond J (2002) 77
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Flower development of Lilium longif
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Flower development of Lilium longif
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"To the people from Brazil, I dedic
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CONTENTS Chapter 1 Introduction: Li
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Chapter 1 Knowing the mechanisms of
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Chapter 1 protein interactions and
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Chapter 1 functionally active (Pela
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Chapter 1 heterologous systems. Thi
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Chapter 1 Although the festiva natu
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Chapter 1 Kramer EM, Irish VF (2000
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Chapter 1 Winter K-U, Weiser C, Kau
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Chapter 2 Introduction Mechanisms o
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Chapter 2 Lily (Lilium longiflorum
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Chapter 2 7 4 4 17 20 30 13 50 26 3
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Chapter 2 inactive in the perianth
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Chapter 2 Flowers derived from plan
Page 34 and 35: Chapter 2 LMADS1 from L. longifloru
Page 36 and 37: Chapter 2 oligo-dT primer. For ampl
Page 38 and 39: Chapter 2 Clough SJ, Bent AF (1998)
Page 40 and 41: Chapter 2 Theissen G, Becker A, Ros
Page 42 and 43: Chapter 3 Introduction Transcriptio
Page 44 and 45: Chapter 3 The ABCDE model is partic
Page 46 and 47: Chapter 3 Results and Discussion Is
Page 48 and 49: Chapter 3 6 23 13 28 22 40 77 52 41
Page 50 and 51: Chapter 3 A B Figure 4. Phenotype o
Page 52 and 53: Chapter 3 Nucleic acids isolation a
Page 54 and 55: Chapter 3 kanamycin. Surviving seed
Page 56 and 57: Chapter 3 Pelaz S, Gustafson-Brown
Page 59 and 60: Ascribing genetic function from lil
Page 61 and 62: + = aaBB AAbb AaBb (wt) if aa + Z =
Page 67 and 68: Discussion Ascribing genetic functi
Page 69 and 70: Cloning of LLAG1 into a binary vect
Page 75 and 76: Transformation of Lilium via partic
Page 79 and 80: lue spots per explant 2.0 1.8 1.6 1
Page 83: Transformation of Lilium via partic
Page 87 and 88: CHAPTER SIX FLORAL HOMEOTIC MUTANTS
Page 89 and 90: A C B D Chapter 6 Figure 1. Floral
Page 91 and 92: Chapter 6 Arabidopsis, giving the e
Page 93 and 94: as visualised in double flowers. Ch
Page 95 and 96: Chapter 6 1997). Additionally, regi
Page 97 and 98: REFERENCES Chapter 6 Byzova MV, Fra
Page 99 and 100: CHAPTER SEVEN Chapter 7 The potenti
Page 101 and 102: Chapter 7 viral vector transcripts
Page 103 and 104: Chapter 7 Guo and Kemphues, in 1995
Page 105 and 106: Initiation phase Maintenance phase
Page 107 and 108: Genes involved in the plant gene si
Page 109 and 110: Chapter 7 sterility due to flower d
Page 111 and 112: Chapter 7 shows the systematic clas
Page 113 and 114: Chapter 7 In tomato, an alternative
Page 115 and 116: Chapter 7 species, in our case part
Page 117 and 118: Chapter 7 functions from monocot ge
Page 119 and 120: Chapter 7 induce phenotypes with ho
Page 121 and 122: Chapter 7 Covey SN, Al-Kaff NS, Lan
Page 123 and 124: Chapter 7 Li QZ, Li XG, Bai SN, Lu
Page 125 and 126: Chapter 7 Tang G, Reinhart BJ, Bart
Page 127 and 128: The ABCDE model for lily EPILOGUE E
Page 129 and 130: Epilogue is an appropriate system a
Page 131 and 132: Epilogue system, such as N. bentham
Page 133 and 134: SUMMARY Summary Lily (Lilium spp.)
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Summary the bialaphos resistance ge
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SAMENVATTING Samenvatting De lelie
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Samenvatting van Arabidopsis thalia
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RESUMO Resumo O lírio (Lilium spp.
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Resumo selvagem de Arabidopsis supe
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ACKNOWLEDGEMENTS Acknowledgements W
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Acknowledgements (two great souls);
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ABOUT THE AUTHOR About the Author V
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