Plant transformation methods

1. Plant transformation methods Dr. Annamalai Muthusamy Manipal Life Sciences Centre Manipal University Manipal – 576 104 Karnataka, India amsamy20@gmail.com

2. Why Plant Transformation Agricultural Production Different goods Plants & Animals

3. Conventional & modern practice Improve the agricultural productivity

4. ICAR Union Minster of Agriculture - President Director – General & Secretary to GOI in DARE 8 Deputy Directors-General Crop Sci, Natural Resource Management, Animal Sci, Agricultural Edu, Agricultural Ext, Fisheries, Horticulture & Agricultural Eng. 33,000 (7000 – Res & Management) in 28 SAUs

5. Plant Agriculture

6. Sustain life on Earth Oxygen Food, Fiber & Shelter Habitats for animals Preserve soil Plants beautify

7. Pulses – macronutrients & minerals, pulses also contain PSMs that are increasingly being recognized for their potential benefits for human health. Health potential of pulses - bioactivity of pulses Isoflavones, phytosterols, resistant starch, bioactive carbohydrates, alkaloids & saponins.

8. Ensure with five food groups Creals Vegetables & fruits Milk & milk products Pulses / non-veg protein Fat, oils & sugars Good breakfast, moderate lunch & light dinner

9. Sufficient, nutritionally adequate & culturally acceptable food for an active, healthy life.

10. Manipulation of Plants

11. Conventional breeding Tissue culture Plants Genetic engineering

12. Conventional breeding ♣ Selection Tissue culture ♣ Without in vitro selection ♣ With in vitro selection

13. Plant Transformation • Plants are the easiest of higher organisms to transform • Both physical and biological methods exist for transformation • Until recently, only transgenic organisms in wide public release were plants

15. Plant Transformation Methods In planta Chemical Biological Physical Microinjection Pressure Biolistics - gene gun/ particle bombardment Electroporation Microinjection Silica/carbon fibers Lazer mediated SAT PEG DEAE-dextran Calcium phosphate Artificial lipids Proteins Dendrimers • A. Tumefaciens • Rhizogenes • Virus-mediated

16. Transformation • Plants - physical methods • Microinjection • Electroporation • Biolistics - gene gun • Silica/carbon fibers • Lazer mediated • SAT

17. Microinjection of GOI

18. This electroporator is for low-current applications such as those using small electrodes

19. Biolistic / Gene Gun

21. Sonication & SAAT

22. Biological Transformation • dsDNA vectors - i.e cassava mosaic virus • Agrobacterium tumefaciens & A. rhizogenes • Soil bacterium • Causes diseases in plants called crown gall & • hairy root • SAAT

23. Biological Transformation ♣ Agrobacteriumtumefaciens & ♣ Agrobacteriumrhizogenes

25. Possible plant compounds, that initiate Agrobacterium to infect plant cells. Acetosyringone, ferulic acid, gallic acid, Hydroxybenzoic acid, pyrogallic acid, vanillin etc. In monocot – not efficient Transformation frequencey – very less

27. Agrobacterium • Agrobacterium(disease symptomology and host range) • radiobacter - “avirulent” species • A. tumefaciens - crown gall disease • rhizogenes - hairy root disease • rubi- cane gall disease • vitis - galls on grape and a few • other plant species Otten et al., 1984

28. Cellular process of Agrobacterium–host interaction Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129

33. Plant Transformation Methods Virus-mediated gene transfer (Plant viruses as vectors) Caulimoviruses – ds DNA – CaMV Geminiviruses - 2ss DNA – maize streak virus RNA plant viruses - TMV

34. In Planta Transformation ♣ Meristem transformation ♣ Floral dip method ♣ Pollen transformation

35. Chloroplast transformation - Horizontal gene transfer

36. Selectable Markers • A gene encoding an enzyme • Antibiotic resistance • Herbicide resistance • Positive selection genes • genes that allow use of some necessary media component.

37. Selectable Markers • NPTII - kanamycin (antibiotic) • Hpt - hygromycin • PMI - changes mannose to useable carbohydrate

40. Novel Selection Genes • Luciferase - gene from fireflies – substrate • Green Fluorescent Protein - from jellyfish - under lights and filter the transgenic plants - GFP • GUS - glucuronidase gene will convert added substrate to blue color.

42. Production of transgenic plants Isolate and clone gene of interest Add DNA segments to initiate or enhance gene expression Add selectable markers Introduce gene construct into plant cells (transformation) Select transformed cells or tissues Regenerate whole plants

43. Synonym - BC-Indicator X-GlcA/ X-Glu X-glucuronideMolecular Formula C14H12BrClNNaO7Molecular Weight 444.59 Activity - quantitative way or through visualization Beta-glucuronidase – E. Coli Richard Anthony Jefferson (1987)

44. X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS GUS oxidative dimerization X-glu → colourless soluble → Blue precipitate of intermediate diX-indigo

45. Chloroplast transformation - Horizontal gene transfer

46. Selection & Regeneration • Cells which contain the selectable marker gene can grow • All plants that develop are transgenic • Plant transformation using physical or biological methods requires a tissue culture stage

47. In Planta Transformation • Meristem transformation • floral dip • Pollen transformation • electroporation

48. Analysis of T0 plants Morphology Physiology Yield characters GUS expression Gene expression Confirmation with selectable marker, Screenable marker, Negative & Positive control

49. Resistance & Stress tolerance in plants: Resistance: - able to break-down or - metabolize foreign molecules or - introduction a new enzyme to metabolize Tolerance: - able to grow -foreign molecules - either the target enzyme or - altered form of enzyme