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UNC-Chapel Hill BIOL 205 - Plant fertilization and transgenic plants

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Plant fertilization and transgenic plantsSlide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Slide 14Slide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 23Slide 24Examples of transgenic plantsSlide 26Slide 27Slide 28Slide 29Slide 30Slide 31Slide 32OOGENESIS IN DROSOPHILASlide 34Slide 35Slide 36Nuclear divisions start without cell division in Drosophila (superficial cleavage)Slide 38Slide 39Slide 40Slide 41Slide 42Slide 43Plant fertilization and transgenic plantsHow does fertilization occur in plants?How do you make a transgenic plant?Flowering plant life cycleMeiosis in female produces four megaspores. One of these makes the gametophyte.Female gametophyte: Megaspore nucleus divides three times to produce 8 nuclei.Then the gametophyte cellularizes.Male gametophyte: pollen grains(vegetative cell)Fertilization:Pollen tube fuses with a synergid.Sperm cells are released.Fertilization: The egg is fertilized to produce the embryo. The central cell is fertilized to produce the triploid endospermembryoendosperm(central cell)ovuletransmitting tractArabidopsis pollination viewed with a pollen-expressed GUS marker line(from Johnson et al., 2004, Genetics 168: 971-982)Pollen tube attraction to Tourenia micropyleHigashiyama and Hamamura (2008) Sexual Plant Reproduction 21: 17-26Pollen tube attraction to Tourenia fournerii micropyle(movie)Higashiyama and Hamamura (2008) Sexual Plant Reproduction 21: 17-26Small peptide applied at time 0(Fluorescent dye allows visualization of possible gradient)(Okuda et al. (2009) Nature 458: 357-361)Small peptides made in the synergids attract pollen(Okuda et al. (2009) Nature 458: 357-361)The peptides are needed to attract pollenKnock down expression of peptide genes in embryo sacsMaize peptides induce pollen tube rupture(Amien et al. (2010) PLoS Biol 8(6): e1000388. doi:10.1371/journal.pbio.1000388)ZmES4 expression pattern:(Amien et al. (2010) PLoS Biol 8(6): e1000388. doi:10.1371/journal.pbio.1000388)Maize peptides induce pollen tube ruptureEffect of ZmES4 knock-down:Wild type Knock down ES4 by RNAi(Amien et al. (2010) PLoS Biol 8(6): e1000388. doi:10.1371/journal.pbio.1000388)Maize peptides induce pollen tube ruptureDouble fertilizationSperm nuclei marked with red fluorophore, female nuclei with green(movie)(Berger et al. (2008) Trends in plant Science 13: 437-443)Making transgenic plantsAgrobacterium tumefaciens causes crown gall tumorsMaking transgenic plantsAgrobacterium-mediated plant transformationkanamycin resistantGene conferring kanamycin resistanceyour favorite geneMaking transgenic plantsRegenerating plants from cultured (transformed) cellsAgrobacterium-mediated Arabidopsis transformationSelect among progeny seeds for transgenic plants (herbicide or antibiotic resistance)Dip Arabidopsis flowers in solution of engineered Agrobacterium(Some female gametophytes are transformed.)Examples of transgenic plantsWidely grown:Herbicide resistantPest resistantOther ideas:Improved nutrition (golden rice, phytase-expressing corn for animal feed)Delayed ripeningSalt tolerancePhytoremediation (decontaminate soils)http://www.isaaa.org/resources/publications/briefs/41/pptslides/Brief41Slides.pdfhttp://www.isaaa.org/resources/publications/briefs/41/pptslides/Brief41Slides.pdfhttp://www.isaaa.org/resources/publications/briefs/41/pptslides/Brief41Slides.pdfhttp://www.isaaa.org/resources/publications/briefs/41/pptslides/Brief41Slides.pdfDrosophila melanogaster developmentHow do Drosophila embryos develop?How can one use genetics to find genes that regulate embryo development?DROSOPHILALIFE CYCLE4 days4 days1 day1 day2 daysLife cycle of Drosophila egglarva1st instarlarva2nd instarlarva3rd instarpupaembryogenesisfemaleOOGENESIS IN DROSOPHILAGermariumring canalGermline Cyst FormationCystoblastPro-Oocyte(undergoes meiosis)germline stem cellsfollicle stem cellsgermline: stem cell > cystoblast > 1 oocyte + 15 nurse cellsNurse cells(undergo enoreduplication)OOGENESIS IN DROSOPHILAGermarium Vitellariumnurse celloocytestalkfollicle cellsborder cellsgermline stem cellsfollicle stem cellsoocyteoocyte + nurse cells surrounded by (somatic) follicle cellsDrosophila oocyte and supporting cellsNurse cells Ring canals Oocyte Follicle cells(from Gonzalez-Reyes and St Johnston (1994) Science 266: 639-642.)Drosophila oocyte and supporting cellsNurse cell nuclei Follicle cell nuclei(from Gonzalez-Reyes and St Johnston (1994) Science 266: 639-642.)Nuclear divisions start without cell division in Drosophila (superficial cleavage)Fig. 9.1Zygotic gene expression beginsegg blastoderm fate maplarvaTALarvae already have substantial patterningacronheadthoraxabdomentelsonT1T2T3A1A2A3A4A5A6A7A8(cuticle) “stripy” expressionof segmentation genefushi tarazu (ftz) anterior posterior ventral dorsal epithelium(6,000 cells)The fruit fly body plan is assembled in 24 hours: How?Christiane Nüsslein-Volhard and Eric Wieschaus used genetics to identify proteins that set up the embryonic body planWieschaus and Nüsslein-Volhardlooked for mutants that affect the fly body plan wildtypeGenes identified in a famous screen for Drosophila mutants with embryo patterning defectsMaternal-effect mutationsGenes expressed during oogenesis (before fertilization)or genes expressed in maternal cells (follicle) All progeny of heterozygous mother are normal.All progeny are affected only if mother is homozygous mutantZygotic mutationsGenes expressed during embryogenesis (after fertilization)Only genetically mutant embryos are affected.(25% of progeny of heterozygous parents are affected.)Drosophila embryonic patterning


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UNC-Chapel Hill BIOL 205 - Plant fertilization and transgenic plants

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