Plant fertilization and transgenic plants How does fertilization occur in plants? How do you make a transgenic plant?

Flowering plant life cycle

Meiosis 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

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 endosperm embryo endosperm (central cell)

ovule transmitting tract Arabidopsis pollination viewed with a pollen- expressed GUS marker line

(from Johnson et al., 2004, Genetics 168: )

Pollen tube attraction to Tourenia micropyle Higashiyama and Hamamura (2008) Sexual Plant Reproduction 21: 17-26

Pollen tube attraction to Tourenia fournerii micropyle (movie) Higashiyama and Hamamura (2008) Sexual Plant Reproduction 21: 17-26

Small peptide applied at time 0 (Fluorescent dye allows visualization of possible gradient) (Okuda et al. (2009) Nature 458: ) Small peptides made in the synergids attract pollen

(Okuda et al. (2009) Nature 458: ) The peptides are needed to attract pollen Knock down expression of peptide genes in embryo sacs

Double fertilization Sperm nuclei marked with red fluorophore, female nuclei with green (movie) (Berger et al. (2008) Trends in plant Science 13: )

Making transgenic plants

Agrobacterium tumefaciens causes crown gall tumors

Making transgenic plants

Agrobacterium-mediated plant transformation kanamycin resistant Gene conferring kanamycin resistance your favorite gene

Making transgenic plants

Regenerating plants from cultured (transformed) cells

Agrobacterium-mediated Arabidopsis transformation Select among progeny seeds for transgenic plants (herbicide or antibiotic resistance) Dip Arabidopsis flowers in solution of engineered Agrobacterium

Examples of transgenic plants Widely grown: Herbicide resistant Pest resistant Other ideas: Improved nutrition (golden rice, phytase- expressing corn for animal feed) Delayed ripening Salt tolerance Phytoremediation (decontaminate soils)

Drosophila melanogaster development How do Drosophila embryos develop? How can one use genetics to find genes that regulate embryo development?

DROSOPHILA LIFE CYCLE 4 days 1 day 2 days Life cycle of Drosophila egg larva 1st instar larva 2nd instar larva 3rd instar pupa embryogenesis female

OOGENESIS IN DROSOPHILA Germarium ring canal Germline Cyst Formation Cystoblast Pro-Oocyte (undergoes meiosis) germline stem cells follicle stem cells germline: stem cell > cystoblast > 1 oocyte + 15 nurse cells

OOGENESIS IN DROSOPHILA GermariumVitellarium nurse cell oocyte stalk follicle cells border cells germline stem cells follicle stem cells oocyte oocyte + nurse cells surrounded by (somatic) follicle cells

Drosophila oocyte and supporting cells Nurse cellsRing canalsOocyte Follicle cells (from Gonzalez-Reyes and St Johnston (1994) Science 266: )

Drosophila oocyte and supporting cells Nurse cell nucleiFollicle cell nuclei (from Gonzalez-Reyes and St Johnston (1994) Science 266: )

Nuclear divisions start without cell division in Drosophila (superficial cleavage) Fig. 9.1 Zygotic gene expression begins

eggblastodermfate map larva T A Larvae already have substantial patterning acron head thorax abdomen telson T1 T2 T3 A1 A2 A3 A4 A5 A6 A7 A8 (cuticle) “stripy” expression of segmentation gene fushi 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 plan

Wieschaus and Nüsslein-Volhard looked for mutants that affect the fly body plan wildtype

Genes identified in a famous screen for Drosophila mutants with embryo patterning defects

Maternal-effect mutations Genes 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 mutant Zygotic mutations Genes expressed during embryogenesis (after fertilization) Only genetically mutant embryos are affected. (25% of progeny of heterozygous mother are affected.) Drosophila embryonic patterning mutants