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Testing Pollen Viability 2006-03-30 Master’s project in the Danish-Swedish Horticulture programme 2006:6 ISSN 1651-1579 Evaluation of transgenic Campanula carpatica plants by Stefan Bengtsson Biology Supervisors Sridevy Sriskandarajah Margrethe Serek Department of Agricultural Sciences The Royal Veterinary and Agricultural University Thorvaldensvej 40 DK-1871 Fredriksberg C Denmark 1 ___________________________________________________________________________ 2 ___________________________________________________________________________ Foreword This Master’s thesis forms part of my studies as a horticulturist at the University of Agricultural Sciences, Sweden. The work was carried out at the Royal Veterinary and Agricultural University, Denmark during the period April - November 2005. I am very happy to have had this opportunity to do my Master’s project in Denmark. I thank all the staff at the Department for support and help, especially since we had difficult circumstances to work under when the labs had to move and so on. The Royal Veterinary and Agricultural University Fredriksberg, March 2006 Stefan Bengtsson 3 ___________________________________________________________________________ 4 ___________________________________________________________________________ Table of contents Sammanfattning ........................................................................................................................... 13 Summary ...................................................................................................................................... 14 General introduction..................................................................................................................... 15 Problem statement ........................................................................................................................ 15 1 Literature review..................................................................................................................... 16 1.1 Campanula species ........................................................................................................ 16 1.1.1 Taxonomy of Campanula carpatica Jecq ......................................................... 16 1.1.2 Description of Campanulaceae family .............................................................. 16 1.1.3 Description of Campanula L. species................................................................ 16 1.1.4 Description of Campanula carpatica Jecq........................................................ 17 1.2 Postharvest quality of ornamentals ............................................................................... 17 1.2.1 Ethylene and postharvest life ............................................................................ 18 1.2.2 Biosynthesis of ethylene.................................................................................... 19 1.2.3 Effects of ethylene on plants and ornamentals.................................................. 21 1.3 Reproduction ................................................................................................................. 22 1.3.1 Development of the flower in Campanulaceae................................................. 22 1.3.2 Pollen viability .................................................................................................. 23 1.3.2.1 Staining for pollen viability................................................................ 23 1.3.2.2 Pollen tube germination ..................................................................... 24 1.3.2.2.1 In vitro germination for pollen viability................................. 24 1.3.2.2.2 In vivo germination for pollen viability.................................. 24 1.3.2.3 Crossing.............................................................................................. 25 1.4 Genetic engineering of ornamentals for longer postharvest life ................................... 26 1.4.1 Campanula......................................................................................................... 27 1.4.2 Carnation ........................................................................................................... 28 1.4.3 Petunia............................................................................................................... 29 1.4.4 Tomato .............................................................................................................. 30 1.4.5 Polymerase chain reaction (PCR) ..................................................................... 30 1.4.6 Promoters .......................................................................................................... 31 1.4.6.1 Cauliflower mosaic virus (35S) as a promoter................................... 31 1.4.6.2 Floral binding protein (fbp) as a promoter ......................................... 32 1.4.7 Gel electrophoresis............................................................................................ 32 1.4.8 Enzyme-linked immunosorbent assay (ELISA)................................................ 33 2 Materials and Methods ........................................................................................................... 35 2.1 Plant material................................................................................................................. 35 2.2 Ethylene sensitivity tests ............................................................................................... 35 2.3 Morphological studies................................................................................................... 37 2.4 Pollen fertility tests........................................................................................................ 37 2.4.1 Aniline blue staining test of pollen ................................................................... 37 2.4.2 Pollen tube germination test.............................................................................. 38 2.4.3 Crossing of transgenic lines .............................................................................. 38 2.5 Molecular investigations ............................................................................................... 40 5 ___________________________________________________________________________ 2.5.1 Extraction of DNA ............................................................................................ 40 2.5.2 Polymerase chain reaction (PCR) ..................................................................... 43 2.5.3 Recipe for the gel .............................................................................................. 44 2.5.4 Neomycin phosphotransferase II enzyme-linked immunosorbent assay (nptII ELISA) ....................................................................................................................... 45 3 Results ...................................................................................................................................... 47 3.1 Ethylene sensitivity tests ............................................................................................... 47 3.2 Morphological studies................................................................................................... 50 3.2.1 Description of the flower organs at different stages ......................................... 50 3.2.2 Description of the different transgenic lines ..................................................... 51 3.3 Results of the pollen fertility tests................................................................................. 53 3.3.1 Results of the aniline blue staining test............................................................. 53 3.3.2 Results of the pollen tube germination test ....................................................... 54 3.3.3 Results of the crossings..................................................................................... 55 3.4 Results from the PCR analyses ..................................................................................... 56 3.4.1 Extraction of DNA ............................................................................................ 56 3.4.2 Results of the first PCR set-up .......................................................................... 56 3.4.3 Results of the second PCR set-up ..................................................................... 58 3.5 Results of the nptII ELISA-test..................................................................................... 59 4 Discussion................................................................................................................................. 61 4.1 Methods......................................................................................................................... 61 4.2 Results ........................................................................................................................... 62 4.2.1 Ethylene sensitivity tests ................................................................................... 62 4.2.2 Morphological studies....................................................................................... 62 4.2.3 Pollen fertility tests............................................................................................ 62 4.2.4 Amplification of etr1-1 using the PCR method ................................................ 63 4.2.5 Concentration of nptII using the ELISA method .............................................. 63 5 Conclusions .............................................................................................................................
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