WELCOME

TOPIC: CREATION OF BT

PLANTS,GOLDEN RICE &FIAVR

SAVR TOMATO

SIJI SKARIAHPG 2 BOTANYST.THOMAS COLLEGEKOZHENCHERRY

Improvement in agricultural production and the food and nutrition situation depends on land water and energy resources.

From 1970 a new type of research started with the aim of producing new varieties of plants by genetic recombination techniques.

They are genetically engineered plants

They have acquired a new trait from the introduced DNA and inherit the trait for many generations.

The new plants produced by such techniques

are supposed to be:-

Virus resistant plant

Insect resistant plants,

Herbicide resistant plant

BACILLUS THURINGIENSIS (BT)

Soil bacterium; ubiquitous

Different strains produce their own insecticide

proteins.

The protein is called as cry protein for its

crystal form.

Each cry protein selectively affects insects

belonging to a particular order

( Lepidoptera(moths,butterflies),Diptera(flies

and mosquitoes) etc)

BT CROPS

Bt plants are generally said that insect resistant

transgenic plants.

Insect attack is a serious agricultural problem

leading to yield losses and reduced product

quality.

Each year, insects destroy about 25 percent

of food crops worldwide.

many transgenic plant with insect

resistantance have been developed by

adopting gene transfer methods.

The gene helps the plants produce proteins that are toxic to certain insects

They reduced the use of chemical pesticides in agriculture

The genetically modified crops is called Bt-crops, because the insect-killing gene in the plant comes from the bacteria Bacillus thuringiensis.

This gene encodes a protein(cry toxin),that is toxic to many insects.

This bacterium is only weakly toxic to insects

as a vegetative cell,but during sporulation.it

produces an intracellular toxic protein

crystal.the parasporal body, that can act as

a microbial insecticide for specific insect

groups

[Lepidoptera(moths,butterflies),Diptera(flies

and mosquitoes) etc].

The parasporal crystal,after exposure to

alkaline conditions in the insect

hindgut,fragments to release the protoxin.

After it react with a protease enzyme,the

active toxin is produced.

Six of the active toxin units integrate into

the plasma membrane to form a

hexagonal shaped pore through out the

mid gut cell.

This leads to the loss of osmotic balance and

ATP and finally to cell lysis.

The insect resistant genetically modified

crops are produced by inserting the Bt

gene(cry gene) from Bacillus thuringiensis

into a crop through genetic engineering

techniques and get the gene expressed.

Such plants can produce the toxic crystal

protein and protect themselves from insects

without any external application of pesticides.

Bt GM crops are protected specifically

against cotton boll worm ,pink bollworm,

colorado potato beetle,European corn

borer,tobacco bud worm etc.

EXAMPLES OF Bt CROPS

Bt cotton

Bt Brinjal

Bt corn

Bt potato

GOLDEN RICE

In 2000,Ingo Potrykus (Swiss Federal

Institute of Technology)and Peter Beyer

(University of Freiburg),created “GOLDEN

RICE”- a trasgenic rice capable of

producing beta carotene, a precursor of

vitamin A in the edible part of

rice,endosperm, by using recombinant

DNA technology.

Deficiency of this vitamin cause night

blindness.

The research was conducted with the aim of producing a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A.

Golden rice was created by transforming rice by the addition of 2 beta carotene biosynthesis genes

1. 2 Daffodil genes(Narcissus pseudonarcissus) -psy(phytoene synthase & phytoene desaturase) and

2. 1 bacterial gene- crt 1 (Lycopene beta cyclase) from the soil bacterium Erwiniauredovora.

The psy and crt 1 genes were transferred into the rice nuclear genome and placed under the control of an endosperm specific promoter.

Hence they are expressed only in the endosperm.

The presence of beta carotene in the rice endosperm gives a characteristic yellow or orange colour to the rice and hence the name “GOLDEN RICE”.

The orginal golden rice was called SGR1

In 2005,a new genetically modified strain of

Golden rice called Golden rice 2,which is

capable of producing 23 times more beta

carotene than the orginal variety,was

produced by combining the phytoene

synthase gene from maize with crt1 gene

from the orginal Golden rice (Biotechnology

Company,Syngenta,U.K)

BIOSYNTHESIS

Biosynthetic pathway of provitamin A is a continuation of Lycopene pathway

The starting point of this pathway is the production of GGDP(Geranyl Geranyl Di Phosphate)

Immature rice endosperm is capable of synthesizing GGDP but subsequent stages not expressed in tissues.

Early transformation with phytoene synthasegene to rice endosperm, specific promoter indicated that phytoene could be synthesized from GGDP in the rice grain.(phytoene is the precursor of beta carotene)

The phytoene is converted into lycopene by phytoene desaturase.

This lycopene is converted to beta carotene by using lycopene beta cyclase.

Flavr Savr Tomato

FLAVR SAVR TOMATO

Flavr Savr Tomato (also known as CGN-

89564-2) is a genetically modified tomato

with slow ripening and improved shelf

life,produced by Calgene Company,

California(1992).

It is the first commercially grown genetically

engineered food to be granted a license for

human consumption.

Fruit softening is promoted by an enzyme Polygalactouronase(PG).

It degrades pectin

In the transgenic Flvr Savr Tomato,the gene that synthesizes Polygalactouronase is blocked and thus prevented it from softening,while still allowing the tomato to retain its natural colour and flavour.

Antisense RNA technology used to develop

Flavr Savr tomato.

Antisense RNA is a single stranded RNA that

is complementary to a messenger

RNA(mRNA).

The novel variety was developed by insertion

of an additional copy of the

polygalactouronase encoding gene in the

antisense orientation, resulting in reduced

translation of the PG messenger RNA.

The PG enzyme is the chief mechanism of

pectin degradation in tomato fruit leading to

fruit softening.

The transgenic variety ripens normally but

experiences less pectin breakdown and,

therefore, has increased thickness and

consistency that benefits all stages of

harvesting and processing.

DEVELOPMENT OF THE MODIFIED PLANT

The FLAVR SAVR™ tomato was created

by Agrobacterium-mediated transformation in

which the transfer-DNA (T-DNA) contained a

copy of the tomato PG encoding gene in the

antisense orientation.

In addition, the T-DNA contained sequences

encoding the enzyme neomycin

phosphotransferase II (NPTII).

The expression of NPTII activity was used as

a selectable trait to screen transformed plants

for the presence of the antisense-PG gene.

Transcription of the antisense-PG gene did not result in the expression of any novel protein.

The antisense PG gene is essentially a reverse copy of part of the native tomato PG gene that supresses the expression of endogenous PG enzyme prior to the onset of fruit ripening.

The mechanism of decreased PG activity in FLAVR SAVR™ tomato is likely linked to the hybridization of antisense and sense mRNA transcripts, resulting in a decreased amount of free positive sense mRNA available for protein translation.

Reduced PG expression decreases the

break down of pectin and leads to fruit with

slowed cell wall breakdown,and delayed

softening.