1. Plant Molecular Genetics and Genetic Transformation HORT 301 – Plant Physiology November 1, 2010 Taiz and Zeiger – Chapter 2, Smith et al. (2010) – Chapter 9 Function of all plant genes – omics; genome, transcriptome, proteome Allelic variation – Le, GA 20 →GA 1 1 Mutations - phenotype

2. Molecular genetics Genetics – study of heredity/inheritance and function of genetic material Molecular genetics – gene identification, structure and function, biotechnology Genotype – genetic constitution, genes Phenotype – appearance and other function(s) resulting from gene function Gene – functional DNA unit, promoter and coding sequence, i.e. locus Griffiths AF, et al. 1996. An Introduction to Genetic Analysis. 6th ed. Locus – usually two alleles at each, contributed by two homologous chromosomes in a diploid (2n) organism, dominant (Y) and recessive (y) alleles yy 2

3. Mutation – change in DNA structure of a gene Mutation may result in loss (reduction in)- or gain-of-function, or may not have an effect Function is inferred based on deviation from normal 3

4. Smith et al. (2010) Plant Biology Fruit size regulated by reduced function of a cell division inhibitor Plant breeders improve crops by transferring “mutations” that confer better phenotypes/traits! Domestication often selects against fitness

5. Gene function based on gain- or loss-of (reduced)-function by transgene expression Dwarfism caused by reduced GA levels, over-expression of GA oxidase 5

6. Vitamin A production by transgene overexpression – trans-kingdom gene transfer 6

7. Smith et al. (2010) Plant Biology Gene (RNA) silencing – natural viral RNA suppression, plant defense 7 Transgene-induced gene silencing – GFP Smith et al. (2010)

8. Determining gene function based on mutagenesis – transgene tagging Forward genetics – phenotype is selected and then the mutation in a specific locus is identified Reverse genetics – mutation in a specific locus is identified and then the phenotype is determined 8

9. Genetic transformation of plants – functional analysis by gene transfer and inheritance of transgene Smith et al. (2010) Plant Biology 9

10. Agrobacterium causes crown gall 10

11. Agrobacterium T-DNA is integrated into the plant genome Tumorization - auxin and cytokinin biosynthesis Tumor growth Opine biosynthesis 11

12. Agrobacterium infection and integration processes 12

13. Smith et al. (2010) Plant Biology “Disarmed” T-DNA vector for plant genetic transformation – elimination of genes that cause tumor formation and growth, opine biosynthesis Binary system includes a vir gene plasmid for replication, infection and integration 13

14. DNA delivery “vehicles” for transformation of plant cells Plant Vector w/Multiple Expression Cassettes Plant Expression Cassette Enhancer/Promoter Coding sequence Terminator/ AAA/3’-UTR Leader 5’-UTR T-DNA is integrated randomly in the plant nuclear genome and is inherited as a single dominant gene (locus) 14

15. Plant regeneration by organogenesis – regenerate plants from individual cells (independent transformation events 15

16. Transformation of sorghum cells and plant regeneration via somatic embryogenesis CONTROL TRANSFORMED 16

17. 4X 35S T-DNA Vector – * pSKI015 Transformation Mutant Plants Plant Genomic DNA T - DNA or Disruption Activation LB 3’-ocs-bar-mas-5’ OriC RB Arabidopsis mutations by T-DNA “tagging” - pSKI015 insertion results in activation or gene disruption 17 T-DNA is inserted randomly into the genome

18. Floral transformation of Arabidopsis 18

19. Generation of T-DNA tagged population of plants Herbicide selection of transformants Propagation and collection of seed 19

20. Root Development (C24) Luciferase Imaging (C24RD29A::LUC) Shoot Development (Col-0 sos3-1) Stress Isolation of NaCl mutants using different approaches Forward genetic approach – phenotypic selection or screening of the T- DNA mutant population 20

21. Identification of the mutated gene – locate the T-DNA insertion (tag) and determine flanking sequence 21

22.  -importin gene – At5g49310 Arabidopsis chromosomes Molecular breeding is a forward genetic approach! 22

23. Reverse genetic approach – determine function of a candidate gene GTL family of putative Ca 2+ /CaM regulated transcription factors involved in stress responses? 23

24. Mutant lines are available to the public Arabidopsis Biological Resource Center (ABRC, www.arabidopsis.org) T-DNA insertion lineswww.arabidopsis.org Genotyping AtGTL1 (At1g33240) gtl1-2 LR L LB L+R L+LB L+LBL+LB no T-DNA- DNA homozygousheterozygous s No T-DNA homozygous heterozygous Select homozygous T-DNA insertion line 24

25. Evaluate phenotype 25

26. Biotechnology Majority of GM crops (corn, cotton, soybeans) are insect or herbicide resistant Virus resistance Pharmaceuticals and nutriceuticals (biofortification) Stress tolerance 26

27. Millions of Hectares of GM Crops 2008 – Total 124 million ha (315A) USA 62.5 Argentina21.0 Brazil15.8 Canada 7.6 India 7.6 China 3.8 Paraguay 2.7 South Africa 1.8 25 countries – 11 industrial and 14 developing Courtesy of Steve Weller 27

28. Courtesy of Steve Weller 28