2. METHODS OF GENE TRANSFER IN PLANTS
Dr. Thirunahari Ugandhar
Assistant Professor in Botany
Govt Degree College Mahabubabad

3. Sufficient, Nutritionally Adequate & Culturally Acceptable Food for an Active, Healthy Life.
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4. Introduction
The “sharing” of DNA among living forms is well documented as a natural phenomenon.
For thousands of years, genes have moved from one organism to another.
For example, Agrobacterium tumefaciens, a soil bacterium known as 'nature's own genetic engineer', has the natural ability to genetically engineer plants.

5. Manipulation of Plants
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6. Conventional breeding Plants Tissue culture Genetic engineering
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7. Why Gene transfer? What is Gene transfer
Insertion of copies of a gene into living cells in order to induce synthes is of the gene's product.
Why Gene transfer?
To add a desired trait to a crop, a foreign gene (transgene) encoding the trait must be inserted into plant cells, along with a “cassette” of additional genetic material. The cassette includes a DNA sequence called a “promoter,” which determines where and when the foreign.
Gene is expressed in the host, and a “marker gene” that allows breeders to determine which plants contain the inserted gene by screening or selection.

8. Why genetically Engineer Plants?
To improve the agricultural, horticultural or ornamental value of a crop plant.
To serve as a living bioreactor for the production of economically important proteins or metabolites
To provide a renewable source of energy
To provide a powerful means for studying the action of genes (and gene products) during development and other biological processes

9. Desired Traits Pest resistance Herbicide tolerance Viral resistance
Drought resistance
Increased nutritional value
Improved Fruit
Altered ripening
Economical benefits: Money is saved on pesticides. Huge money upfront, but with a shorter amount of time to produce desired product, preciseness, and no unwanted genes.
Herbicide-resistance: GM crops can be produced to be herbicide resistant . This means farmers could spray these crops with herbicide and kill the weeds, without affecting the crop. IN effect, the amount of herbicide used in one season would be reduced, with a subsequent reduction in costs. In some cases pesticide use is not needed, which saves money and protects the environment. (experimenting to make drought or salt-tolerant or less reliance on fertilizer)
Better quality foods-Animals can be genetically modified to be leaner, grow faster, and need less food.

10. Genetic Engineering
Genetic engineering, also called genetic modification, is the direct manipulation of an organism's Genome using Biotechnology
New DNA may be inserted in the host genome by first isolating and copying the genetic material of interest using Molecular Cloning methods to generate a DNA sequence, or by synthesizing the DNA, and then inserting this construct into the host organism.

11. Engineer
An Engineer is a Professional Practitioner of Engineering, concerned with applying Scientific Knowledge and ingenuity to develop solutions for technical problems.
Engineers design materials, structures, and systems while considering the limitations imposed by practicality, regulation, safety, and cost.
The word Engineer is derived from the Latin roots ingeniare ("to contrive, devise") and ingenium ("cleverness")

12. TRANSGENIC PLANTS
The process of introducing new genes into plants to produce new traits is called Plant Genetic Engineering.
The product of this process is called Transgenic Plants. Transgenic plants contain a gene or genes which have been artificially inserted. The inserted genes are known as transgenic.
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13. TRANSGENIC PLANTS – AN INTRODUCTION
Transformation – The process of obtaining transgenic plants.
Transgenic plant – a plant with a foreign gene (or genes) from another plant/animal that is incorporated into its chromosome
Marc Van Montagu and Jeff Schell, discovered the gene transfer mechanism between Agrobacterium and plants, which resulted in the development of methods to alter the bacterium into an efficient delivery system for genetic engineering in plants.
Most common genes (and traits) in transgenic or biotech crops herbicide resistance Insecticide resistance, Bt genes in field corn (maize) virus-resistance (coat-protein) genes

14. WHY HAVE GM CROPS? Growing Human Population Loss of Farmable Land
Remediation of Soil
Enrich Nutrient Content
Transgenic
Wild-type

15. Plant Transformation Methods
Physical
Chemical
Biological
In planta
Microinjection
Pressure
Biolistics - gene gun/
particle bombardment
Electroporation
Silica/carbon fibers
Lazer mediated
SAT
PEG
DEAE-dextran
Calcium phosphate
Artificial lipids
Proteins
Dendrimers
A. Tumefaciens
Rhizogenes
Virus-mediated
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18. Genetic Transformation
PLANTS - PHYSICAL METHODS
Microinjection
Electroporation
Biolistics - gene gun
Silica/carbon fibers
Lazer mediated
SAT
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19. 1. MICROINJECTION
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20. 2. Electroporation
This electroporator is for low-current applications such as those using small electrodes
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21. 3. Biolistic / Gene Gun
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22. 3. Biolistic / Gene Gun

23. 4. SILICA/CARBON FIBERS

25. Streptothricin Acetyl Transferase (SAT)
TRANSFORMATION
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26. Chemical Gene Transfer
PEG
DEAE-dextran
Calcium phosphate
Artificial lipids
Proteins
Dendrimers

27. 1. PEG Gene Transfer

28. 2. Dead Dextran Mediated Gene Transfer

29. 3. Calcium phosphate Transfer

30. Artificial lipoplex Mediated Gene Transfer

32. Biological Transformation
Agrobacterium tumefaciens &
Agrobacterium rhizogenes
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34. BIOLOGICAL TRANSFORMATION
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
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36. 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
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Otten et al., 1984

37. Cellular process of Agrobacterium–host interaction
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3),
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41. Plant Transformation Methods
Virus-mediated gene transfer
(Plant viruses as vectors)
Caulimo viruses – ds DNA – CaMV
Gemini viruses - 2ss DNA – maize streak virus
RNA plant viruses - TMV
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43. POLYMERASE CHAIN REACTION
PCR (polymerase chain reaction) is a method to analyze a short sequence of DNA (or RNA) even in samples containing only minute quantities of DNA or RNA.
 Kary Banks Mullis (Born December 28, 1944) is a Nobel Prize -winning American Biochemist, author, and lecturer. In recognition of his improvement of the Polymerase Chain Reaction (PCR) technique

44. PCR Animation Denaturation: DNA melts Annealing: Primers bind
This is an animation of one step in the PCR process. Take a few minutes and let the animation run through a number of times. It will recycle on its own. This step will show the denaturation (converting the DNA from single- to double-stranded state). The second step is annealing (the binding of the primer to the single-stranded DNA). The final step is extension (the duplication of a strand from the end of the primer).
Denaturation: DNA melts
Annealing: Primers bind
Extension: DNA is replicated

45. Recombinant DNA & Plasmids Combining genes from different sources and/or species
Circular DNA from bacteria = plasmids
Target DNA recombined in plasmid
Bacteria rapidly reproduces many clones

46. Gene transfer techniques…

47. 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
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48. 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
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50. Which foods contain GM Product?
India Approval for GM food crops
Corn
Soy
Papaya
Canola
Potato
Chicory
Rice
Squash
Sugarbeet
Tomatoes
Approval does not necessarily
mean these crops are distributed
Database of GM crops:
Bolded crops are widely grown & sold in US.

51. Genetically Engineered Foods
India approval for GM foods:
Corn
Soy
Papaya
Canola
Potato
Rice
Tomatoes
India approval does not mean that these crops are distributed.
20% of India grocery food contains GM ingredients
Some current trials: apples that resist insect attack, bananas free of viruses and worm parasites, coffee with a lower caffeine content, cabbage that resists caterpillar attacks, melons that have a longer shelf life, sunflowers that produce oil with lower saturated fat.

52. Molecular agriculture makes new gene combinations possible
Peas (on the left) that make a genetically engineered bean protein are insect-resistant and do not need to be sprayed with pesticides.

53. Molecular agriculture makes new gene combinations possible
Peas (on the left) that make a genetically engineered bean protein are insect-resistant and do not need to be sprayed with pesticides.

54. Some GM crops will improve the nutritional quality of foods
Some GM crops will improve the nutritional quality of foods. Such foods are now in India.

55. The approach is to down regulate the expression of the gene encoding the major allergenic protein (antisense)

56. Truthful labels can be misleading or meaningless
There are no GM apples anywhere!
Is food labeled this way
nutritious?

57. How to Label? GMO Conventionally grown
Conventional grown
GMO
Conventionally grown
Pesticides, twice a week Pesticide free

58. Genetic change resulting from crop domestication took 10,000 years.
Teosinte (top) and corn or maize (bottom)

60. Genetically modified crops in future
Cauliflower
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61. Purple tomatoes high in anthocyanins
High anthocyanin purple tomato and red wild-type tomato
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62. Conclusion
Consumer fears are all potential risks, some more than others, and use of GM crops should only be continued with extreme care and intense long term research on the topic should be continued. With most of the cases the use of GM crops can only be justified when the conventional methods are worse and pose even higher risks to the environment.
Also, the labeling that is enforced in much of the world should also be mandatory here in the United States.

63. Transgenic and Nontransgenic Crops
Global Area (mil ha)
Transgenic (mil ha)
% Transgenic
Soybean 91
58.6 64
Cotton 35
13.4 38
Canola 27
4.8 18
Maize
148
25.2 17
Total
301
102 34
James, 2006

64. Prof. N. Rama swamy
My grateful acknowledgement is due to Prof. N. Rama swamy (Teacher of my supervisor) Head Department of Bio-technology, University PG College Kakatiya University Warangal for their kind encouragement and blessing.
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65. tugandharbiotech@gmail.comin