Plant transformation methods. Dr. Annamalai Muthusamy Manipal Life Sciences Centre Manipal University Manipal – 576 104 Karnataka, India email@example.com. Why Plant Transformation. Agricultural Production Different goods Plants & Animals. Conventional & modern practice
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Dr. Annamalai Muthusamy
Manipal Life Sciences Centre
Manipal – 576 104
Plants & Animals
Improve the agricultural productivity
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
Food, Fiber & Shelter
Habitats for animals
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.
Vegetables & fruits
Milk & milk products
Pulses / non-veg protein
Fat, oils & sugars
Good breakfast, moderate lunch & light dinner
Sufficient, nutritionally adequate & culturally acceptable food for an active, healthy life.
♣ Without in vitro selection
♣ With in vitro selection
Biolistics - gene gun/
This electroporator is for low-current applications such as those using small electrodes
♣ Agrobacteriumtumefaciens &
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
Otten et al., 1984
Tzvi Tzfira and Vitaly Citovsky, 2002, Trends in Cell Biol. 12(3), 121-129
Virus-mediated gene transfer
(Plant viruses as vectors)
Caulimoviruses – ds DNA – CaMV
Geminiviruses - 2ss DNA – maize streak virus
RNA plant viruses - TMV
♣ Meristem transformation
♣ Floral dip method
♣ Pollen transformation
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
X-Glu X-glucuronideMolecular Formula
Activity - quantitative way or through visualization
Beta-glucuronidase – E. Coli
Richard Anthony Jefferson (1987)
X-glcA (X-gluc or X-glc or X-glcU) - substrate for GUS
X-glu → colourless soluble → Blue precipitate of
Confirmation with selectable marker,
Screenable marker, Negative &
- able to break-down or
- metabolize foreign molecules or
- introduction a new enzyme to metabolize
- able to grow -foreign molecules
- either the target enzyme or
- altered form of enzyme
Traditional rice is white (a).
The prototype of golden rice was developed in 2000 and is a light yellow
color (b). It contains 1.6 mg/g of carotenoid.
In 2005, new transgenic lines were developed that dramatically increased the amount of carotenoid synthesized, making the rice a deep golden color (c).
This latest form contains 37 mg/g of carotenoid, of which 84% is b-carotene – trial
Miraculin - taste-modifying protein – miracle fruit, the red berries of Richadella dulcifica - shrub native to West Africa
Active principle - protein miraculin - not sweet
Unusual property - turn a sour taste into a sweet taste
Sour foods - lemons, limes & grapefruit, taste sweet when tasted together with this protein
leaves (102.5) &
Fruits(90.7) μg/g fresh weight
Tomatoes comes in many varieties, colors and shapes
Transgenic tomatoes - expressing different malarial antigens
Normal and mutant tomato fruit
high-pigment 1 (hp1/hp1), high-pigment 2 (hp2/hp2), Never-ripe (Nr/Nr),
Green-ripe (Gr/Gr), Colorless non-ripening (Cnr/Cnr) &
ripening-inhibitor (rin/rin) mutations
Transgenic corn kernels (a)
Corn snack (b) or
Embryo or germ cells (c)
Crop & Food Research in New Zealand and his collaborators in Japan
As onions are sliced, cells are broken, alliinases - break down aa sulphoxides - generate sulphenic acids - unstable - rearrange into a volatile gas - syn-propanethial-S-oxide – diffuses by air - reaches the eye - reacts with the water to form a diluted solution of sulphuric acid - Tear glands produce tears to dilute and flush out the irritant
High anthocyanin purple tomato and red wild-type tomato
Prof Cathie Martin from the John Innes Centre Anthocyanins offer protection against certain cancers, cardiovascular disease and age-related degenerative diseases. Anthocyanins also have anti-inflammatory activity, promote visual acuity and hinder obesity and diabetes.Tomatoes already contain high levels of the antioxidant lycopene. Highly processed tomatoes are the best source, or tomatoes cooked in a little oil, which helps to release the lycopene from cells.
Flavonoids meanwhile are soluble in water, and foods containing both water soluble and fat-dissolved antioxidants are considered to offer the best protection against disease. In this study the scientists expressed two genes from snapdragon that induce the production of anthocyanins in snapdragon flowers. The genes were turned on in tomato fruit.
Anthocyanins accumulated in tomatoes at higher levels than anything previously reported for metabolic engineering in both the peel and flesh of the fruit. The fruit are an intense purple colour.
Aresa – Danish biotech company
- planting tg tabacco plant to detect
- Permission from Serbian authorities
- Enzymatic detection & destruction
19 strains of Rhodoccus – use RDX as N2 source
Cytochrome p450 system - breakdown
World's First Blue Roses On Display In Japan- Danielle Demetriou, Daily Telegraph, October 31, 2008, See the rose at http://www.telegraph.co.uk/news/worldnews/asia/japan/3327043/Worlds-first-blue-roses-on-display-in-Japan.htmlTokyo, Japan - World's first blue roses have been unveiled to the public for the first time at an international flower fair in Japan, following nearly two decades of scientific research. The blue-hued blooms are genetically modified and have been implanted with a gene that simulates the synthesis of blue pigment in pansies.
Its scientists successfully pioneered implanting into the flowers the gene that produces Delphinidin, the primary plant pigment that produces a blue hue but is not found naturally in roses.
The world's first genetically modified blue roses were unveiled in the laboratory four years ago, although further research was required to make them safe to grow in nature.
The Blue Rose was developed by Suntory Flowers
Bozeman, Mont. -- U.S. scientists say a FUNGUS in the Patagonian rainforest might be a new source of biofuels since it produces a number of diesel compounds from cellulose.
"This is the only organism that has ever been shown to produce such an important combination of fuel substances," said Montana State University Professor Gary Strobel, making it a better source of biofuels than anything used now.
The fungus, Gliocladium roseum, produces various molecules made of hydrogen and carbon that are found in diesel, the researchers said. Because of that, the fuel it produces is called "myco-diesel."
"Gliocladium roseum lives inside the Ulmo tree in the Patagonian rainforest," Strobel said. "We were trying to discover totally novel fungi in this tree by exposing its tissues to the volatile ANTIBIOTICS of the fungus Muscodor albus. Quite unexpectedly, G. roseum grew in the presence of these gases when almost all other fungi were killed.
"It was also making volatile ANTIBIOTICS. Then when we examined the gas composition of G. roseum, we were totally surprised to learn it was making a plethora of hydrocarbons and hydrocarbon derivatives," Strobel said."
Strobel said the discovery brings into question scientists' knowledge of the way fossil fuels are made.
The discovery is reported in the journal Microbiology.
The fungus grows inside the Ulmo tree in the temperate Patagonian rainforest of Chile and Argentina.
Utrecht University (The Netherlands) and Ghent University (Belgium) with help from scientists in Japan, USA and Switzerland. The results of this research appeared as an advance online publication of the weekly science journal Nature on 26 October 2008.
Pankaj Dhonukshe discovered a molecular switch to alter the auxin transport.
By turning on the switch, Dhonukshe was able to reduce the extent of auxin transport towards the roots.
The hormone then began to accumulate at the places in the young leaves where it is produced and roots began to emerge here where normally leaves would grow.
The photo on the left shows a normal plant with normal leaves and a root and the photo on the right shows a plant on which root has started to grow at the place of young leaf. The shoot part is shown in orange and the roots in green.