1. MATIULLAH(Ph.D)
11-ARID-3967
Date:18:12:2015

2. GENETIC ENGINEERING AND THE STORY OF “GOLDEN RICE”

3. OBJECTIVES What are genetically modified rice?
Why the rice are modified?
How genetic modifications be done?
Is it safe to grow and eat genetically modified rice?
What are the issues surrounding Golden rice?
What are advantages and disadvantages of golden rice?
Why is it not a large scale distribution?
What is the next step?

4. CREATION Beginning of 21st century.
124 million people in 118 countries.
In Africa and South East Asia.
Affected by Vitamin-A deficiency.
Project started in 1992 and publication in
By Ingo Potrykus and Beyer.
A significant break through in Biotechnology.

5. VITAMIN A DEFICIENCY Weakens the immune system. Lead to blindness.
Influence severity of diarrhea and measles.
Increases the risk of death.

6. Genetically modified rice
Fig
Genetically modified rice
Figure “Golden Rice” and prevention of blindness associated with vitamin A deficiency
Ordinary rice

7. Percentage of stunted children

8. THE PROBLEM
400 million poor in rice based societies are Vitamin A deficient.
500,000 children per year go blind.
1.15 millions VAD-precipitated deaths among children world wide.
Rice is the staple food for these but it lack pro-vitamin A.

9. Rice in the Asian Diet

10. Estimated Annual Number of Child Deaths Precipitated by Vitamin A Deficiency (Asia)

11. What is Golden Rice? GM rice by GE. Variety of Oryza sativa.
Designed to produce beta-carotene.
Reason for golden color.
Precursor of Vitamin A.
Part of rice we eat is Endosperm.
Carotenoid pigment present in leaves.
Endosperm devoid of it.

12. Genetic Engineering?
Genetic engineering is the basic tool set of biotechnology
Genetic engineering involves:
Isolating genes
Modifying genes so they function better
Preparing genes to be inserted into a new species
Developing transgenes
Genetic engineering is the collection of techniques necessary to create a transgene. These procedures include isolating the gene-of-interest from the tens of thousands of genes found in the genome of a species. Once that gene is isolated, it is modified so it functions better in an organism. That gene is then mixed with other genes to prepare it to be introduced into another organism. This whole step develops transgenes.

13. WHY GENETIC ENGINEERING?
Resistant varieties.
Improved nutritional value in different food products.
Improved aroma, taste and structure of agricultural products
Increased amount of vitamins in products (golden rice – provitamin A)

14. SCIENCE BEHIND THE GOLDEN RICE
Rice created by transforming rice with two beta-carotene biosynthesis genes:
psy (phytoene synthase) from daffodil (Narcissus pseudo narcissus)
Crt1(PHYTONE DESATURASE) from the soil bacterium Erwinia uredovora

15. Psy and crt1 transformed into rice nuclear genome
Under the control of an endosperm-specific promoter.
Only expressed in the endosperm.
The exogenous lyc gene has a transit peptide sequence.
It is targeted to the plastid.
where geranylgeranyl diphosphate formation occurs.
crt1 gene can catalyze multiple steps in the synthesis of carotenoids
The end product of the engineered pathway is lycopene.
Plant's endogenous enzymes process the lycopene to beta-carotene

16. Lycopene-beta-cyclase
-Carotene Pathway Problem in Plants
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Problem:
Rice lacks
these enzymes
Unlike the single-step RoundUp Ready pathway, the β–carotene synthesis pathway involves multiple enzymes. This important vitamin A precursor cannot be synthsized in rice because it lacks four of the key enzymes. Therefore, the precursor is not made, and the plant contains white kernels.
Normal
Vitamin A
“Deficient”
Rice

17. The Golden Rice Solution
-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Phytoene
Lycopene
 -carotene
(vitamin A precursor)
Phytoene synthase
Phytoene desaturase
Lycopene-beta-cyclase
ξ-carotene desaturase
Vitamin A
Pathway
is complete
and functional
Daffodil gene
Single bacterial gene;
performs both functions
In a major feat of genetic engineering, scientists inserted a complete functioning -carotene biosynthetic pathway into the rice plant. They did this by inserting genes from daffodil the produce functioniong versions of the first and last enzymes of the pathway. In addition, a single bacterial gene that provides the same function as the second and third enzymes of the pathway, was also introduced. With a functioning pathway, the transgenic rice is able to produce the vitamin A precursor β-carotene. It is this product that gives "Golden Rice" its characteristic yellow color.
Daffodil gene
Golden
Rice

18. Developing Transgenics
Introducing the Gene or
Developing Transgenics
Steps
1. Create transformation cassette
2. Introduce and select for transformants
It is now time to cover the development of transgenic crops in greater depth. The two major steps are creating a transformation cassette that contains the gene of interest, and then successfully introducing the cassette into the plant.

19. Gene construct used to generate Golden Rice.
RB, T-DNA right border sequence; Glu, rice endosperm-specific glutelin promoter; tpSSU, pea ribulose bis-phosphate carboxylase small subunit transit peptide for chloroplast localisation; nos, nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus pseudonarcissus (GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi, phosphomannose isomerase's gene from E. coli for positive selection (GR2); LB, T-DNA left border sequence.

20. Distinguishes transformed an un transformed plants
Transformation Cassettes
Contains
1. Gene of interest
The coding region and its controlling elements
2. Selectable marker
Distinguishes transformed an un transformed plants
All transformation cassettes contain three regions. The “gene of interest” region contains the actual gene that is being introduced into the plant. This is the gene that provides the new function to the plant. In this diagram, the region is shown in red.
Many plant tissues are treated with the transformation cassette during the transformation step. Not all of these tissues actually receive the cassette. To distinguish those that contain the gene from those that don’t, it is necessary to use a selection process. The selectable marker is a gene that provides the ability to distinguish transformed from non-transformed plants. This is shown by green.
The most common method to introduce the transformation cassette is by using the plant pathogen Agrobacterium. For this system to work it is necessary that the cassette contain insertion sequences that are used by the bacteria. These are shown by the gray.
3. Insertion sequences
Aids Agrobacterium insertion

21. Prepare tissue for transformation
Transformation Steps
Prepare tissue for transformation
Leaf, germinating seed, immature embryos
Tissue must be capable of developing into normal plants
Introduce DNA
Agrobacterium or gene gun
culture plant tissue
Develop shoots
Root the shoots
This slide summarizes the steps necessary for plant transformation.
Field test the plants
Multiple sites, multiple years

22. Two major delivery methods
Delivering the Gene
to the Plant
Transformation cassettes are developed in the lab
They are then introduced into a plant
Two major delivery methods
Agrobacterium
Two techniques are used to deliver DNA found in the transformation cassette into plant tissues during the plant transformation process. One is a biological system based on the plant pathogen Agrobacterium tumefaciens. The second is a mechanical method where the DNA is “shot” into plant cells using a gene gun.
Regardless of the delivery method, the delivery system must use a plant tissue source that can be manipulated to produce new plants.
Tissue culture
required to generate
transgenic plants
Gene Gun

23. The Lab Steps
And this slide illustrates those steps.

24. Herbicide Resistance Non-transgenics Transgenics
The Next Test Is The Field
Herbicide Resistance
Non-transgenics
The last step in plant genetic engineering is field testing. This slide shows a field that contains herbicide resistant and tolerant plants.
Transgenics

25. 1ST AND 2ND GENERATION GOLDEN RICE
1ST GENERATION:
The original golden rice called SGR1
Produces1.6 µg/g of carotenoids.
2ND GENERATION:
In 2005, a team of researchers at Syngenta, produced "Golden Rice 2“
By replacing genes with homologues from other sources or modifying their regulatory regions.
Produces 23 times more carotenoids up to 37 µg/g

26. WHY GOLDEN RICE HASN'T BEEN APPROVED FOR CONSUMPTION
AROUND THE WORLD??

27. OPPOSITIONS Critics of GM crops have raised various issues: Social
Political
Environmental issues

28. A Dangerous Experiment
In Feb international scientists:
In adequately described in terms of biochemical make up
Not been shown stable over time.
No approval process all over the world.
Open the door to more widespread use of GMOs.

29. Not proven effective
Rice not containing vitamin-A but its precursor beta carotene.
No data published on bioavailability and conversion of beta carotene into vitamin-A

30. Not proven safe to eat Absence of animal testing data.
Retinoids causes birth defects.
Hypervitaminosis.
Unexpected toxins and allergens may arise.

31. Not proven safe for environment
Rice will cross pollinate with other cultivated rice.
Loss of biodiversity in food crops.

32. Better alternatives are available
It encourages a diet based on single industrial staple food.
WHO describes alternatives:
Promoting breast feeding
High dose vitamin-A supplements
Food fortification
Cultivating the garden

33. Organization of the Golden Rice Network
Philippines: International Rice Research Institute, National Rice Research Institute (PhilRice)
Vietnam: Cuu Long Delta Rice Research Institute
Germany: University of Freiburg
India: Department of Biotechnology India, Directorate of Rice Research, Indian Agricultural Research Institute, University of Delhi - South Campus, Tamil Nadu Agricultural University, Agricultural University Pantnagar, University of Agricultural Sciences Bangalore, Chinsurah Rice Research Station
Bangladesh: Bangladesh Rice Research Institute
China: Huazhong Agricultural University, Chinese Academy of Sciences, Yunnan Academy of Agricultural Sciences
Indonesia: Agency for Agricultural Research & Development

34. IN PAKISTAN Farmers say NO! to GM rice.
Missed the opportunities during the Green Revolution.
Serious lack of appreciation of biotechnology at the public and industrial levels
There is need of resource center.
Efforts to develop:
Network of institutions and
individuals working in this field.
BIOTECHNOLOGISTS in NIBGE, NCEMB, NIAB, CCRI, PU, Faisalabad agriculture uni etc.

35. CONCLUSIONS It’s totally Humanitarian project.
Narrow view of technology is incorrect.
The trait will be made available free of cost.
There is need to promote GE.

36. REFERENCES
Potrykus, I Golden Rice and Beyond. Plant Physiology 125:
UNICEF. Vitamin A deficiency
Tang G et al (2009) Golden Rice is an effective source of vitamin A Am J Clin Nutr Jun;89(6): Epub 2009 Apr 15, Retrieved September 6, 2010
Shiva, V. The Golden Rice Hoax
Friends of the Earth. Golden Rice and Vitamin A Deficiency.

37. THANKS