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Production of insecticidal transgenic crop with novel variety of

Vegetative Insecticidal Proteins; by using  as a selectable

marker.

INTODUCTION:

Year by year population of the world is growing dramatically, for instance in 1960,s Indian

population is about 44 millions, in 2010 it reaches to 120 million, almost it increases to

63% on contrary to this the food production increased by only 42%, this too much

variation shows food insecurity [Poverty (Food Insecurity) is the most kind of Violence-

Mahatma Gandhi]. This variation is mainly due to effects of insect pests, natural calamity,

climatic changes, industrialization etc. Among those majority of crop lose is due to

insectpest, ever year almost 30-40% crop lose is by different pest species like lepidoperan,

spodopteran, coleopterans. To decrease the effect of insetpest on crops from last two-three

decades farmers are using synthetic pesticides and insecticides. But this insecticides and

pesticides are not fully bio degradable and even entering into food chain eventually leading

into biomagnifications. To overcome this there come a concept called GM crops for

insecticidal resistance. To develop insecticidal transgenic crops a Gene of Interest and

Selectable marker genes introduced under a specific promoter into interested crops.
[ITERATURE SURVEY:

Selectable markers: are being used in identify tissue/plants having our gene of interest.

Although many selectable markers are available each of them has their own constraints or

negative impacts on environment. Most frequently used selectable markers includes

antibiotic and herbicide resistant genes, they are a  


 genes. 
 genes

confer resistance to phosphinanthracin which is active ingredient of herbicides like BASTA,

liberty, herbiace and bilaphos.   gene confers resistance towards herbicide

glyphosate. There is a possibility of horizontal gene transfer of herbicide resistant genes to

its wild varieties leading to producing Ǯǯsuper weedǯǯ which could be hard to control by

available herbicides. Antibiotic resistance genes like a    which are resistance to

neomycine and hygromycine respectively are also having a chance of horizontal gene

transfer to gut or environmental bacteria, rendering less susceptible to antibiotics. Till date

there is no healthy/environmental risks have been identified by usage of antibiotic or

herbicidal resistant genes. Yet there is a large public concern about usage of antibiotic or

herbicidal resistant genes in plant systems. In order to fulfill this, scientists are trying to

develop alternatives; in the process as of now they developed marker free technologies and

eco friendly markers.

Marker free technologies include [1] site specific recombination system by the use of 

, R/Rs genes. [2] Intra genomic relocation of Trans genes by use of 
 Transposable

elements. [3] D-Amino acid metabolism by doa-1 gene which encodes D- amino acid

oxidase.
Eco friendly markers: this includes Phospho Mannose Isomerase( ) enzyme derived

from a gene of    gene is very common in nature, found in across the

kingdoms, although less so in plant kingdom, the enzyme has been reported to be present

in soybeans and several other legumes, but absent in several other plants (Gold sworthy

and Street., 1965; [ee and Matheson, 1984). Therefore we can successfully use this a

gene as a selectable marker in plant species which are actually deprived of natural 

enzyme. under control of plant promoter allows plant cells to utilize mannose as a

source of carbon and survived on media containing mannose. Non tranformants are out

grown by the deprive of carbon source (Biosen .1994; Joersbo .1998).

Advantage of  as a selectable marker over other markers:

1.Y Non toxic therefore public will not more concern about usage.

2.Y End product (Mannose -6-Phosphate) is harmless, use full in plant metabolism as a

carbon source.

3.Y Non transformed cells are outgrown by deprive of carbon source rather than killing

by herbicides /antibiotics.

4.Y HGT is rare, if at happens, it occurs between

iY Plant to gut bacteria (most of bacteriaǯs are having a gene)

iY Plant to animal cells(all EK are having these gene)

iY Plan to other species or same species plants(need not worry because it

enables uptake of mannose as a extra carbon source)

5.Y PMI acts as a both scorable marker (gene expression assay), as well as selectable

marker.
Gene expression assay: Transforments can be detected by simple Chloro Phenol Red assay

(CPR) in early stages of transformation. In CPR assay multiwell plates are filled with

medium+mannose+CPR followed by a piece of plant tissue. This plant tissues utilizes

mannose in medium and acidifies, acidic conditions favor initial red color of CPR to yellow

color.

6.Y CPR assay reduces number of plantlets send for PCR analysis. [iterature shows

results of CPR assay and PCR was nearly 100% (wringlt etal., 2001).

7.Y We can use thisa gene as a new marker for identification of traits.

8.Y Transformation frequency is high when ais used instead of bar gene in duram

wheat (Gadal et al.2006)and in maiz(Janet et al.2001)

9.Y safety concern: (Syngenta seeds Inc, USA) The  protein, gene is vigorously tested

in concern of safety in different dimensions like

iY Allergenicity evaluation by comparing with known allergens.

iY Glycoprotein profile study of non transgenic plants with transgenic plants.

iY Pleiotrophic effect, toxicity analysis.

iY Study of different agronomic characters.

Therefore use ofagene as a selectable marker is very much good over others.
Gene of interest: Insecticidal proteins

From last couple of decades a number of new GM Crops have been produced and

cultivating successful throughout world. In USA alone some 10-12 crops are cultivating, R;

soy, maiz, cotton, barley etc. most of GM crops are produced for an important agronomic

character called insect resistance (hereafter referred as insecticidal resistant transgenic

crops).in production of insecticidal resistant transgenic crops scientist used 

genes(From  aa) particularly  . These two genes are most

successful against a number of lepidopteron species, some extent to spodoptera and

coleopteran species. These two genes are patented by Monsanto Company, USA.

As far as coming to Indian context only one GM crop that is cotton is now in market

which was launched officially in the year 2002 by Monsanto Ȃ Mhycho bilateral

collaboration with the permission of Indian government. Statistics show that, since the

introduction of transgenic cotton, the area under cotton cultivation has sturdily increased

from 7.7mha in 2002 to 9.4mha in 2008. Production has also increased from 2.3mt in 2002

to 5.4mt in 2008 with the jump of productivity from 302 to 561 kg/ha (Asian-pacific

consortium on agriculture biotechnology.2009). Pesticide consumption is almost 50%

declined after release of Transgenic Bt cotton due to reduction in number of applications

against insects (Subraminian .2009).

But recent publications show that most promonant bollworm Helicoverpa armigera is

surviving on Bt-1 andBt-2 crops,the survivings not only completing their life cycles but also

reproducing successfully(M.T.Ranjit,A.Prabhuraj. November 2001). In November 2009

Monsanto scientists found that the pink bollwarm had become resistant to  cotton in
parts of Gujarat India. In four regions, Amreli, Bhavnagar, Junagarh and Rajkot the -crop

is no longer effective at killing the pests. This was the first instance of resistance that

was confirmed by Monsanto anywhere in the world. The reason for getting resistance is

may be due to considerable variation in the quantum of toxins produced in different parts

of plants and different times of growing season. Such variation has been speculated to

cause differential survival of target insects which gradually leads to resistance in insects.

This indicates that in very near future there is a possibility of discovering potential

resistant population ofR   in feilds towards  toxins. Apart from this

in india several research labs and universities are producing insect resistant transgenic

plants by using the same  and  genes, R; castor ,Sorghum, Green gram,

Ground nut, Black gram etc. although they are in field trails in near future we may see some

of the crops in market. If once lepidopteron species gets resistance towards this  genes

there will be no use of releasing this new  crops in to market which eventually leads to lot

of public money gone to be waste. In view of this we must aware farmers in Refugiea

maintenance which will slow down the process of getting resistance of insects. Apart from

this we must also thrive for alternatives like novel insecticidal proteins, those are

Vegetative insecticidal proteins (VIPs) and Cholesterol Oxidase which have broad host

insect spectra. This VIPs are produced during vegetative cycle as well as in sporulation

form o  aaand  . There are three classes of VIPs R: VIP-1, 2, 3.

VIP1, 2 are toxic to agricultural impotant insects like corn root worm (Jun Fang, Ping

Wang.2007). VIP-3 are very toxic against a range of lepidopteron pests and some of

spodopteran insects(Juan J.Estruch.1996) and their action remains same as like Cry

proteins but their receptors are different. Therefore VIP3s are good candidates for
resistant management strtagies involving stacking or rotation of proteins with different

insecticidal mechanisms. These VIP3s are again subcategorised based on their gene

sequence and three dimensional folding. In recent years integration of this VIP3Aa1 into

R aeventually enhances the insect hosta spectra (Yin Quin, Sheng-Hau Ying,

May 2010). [iterature show that this VIPs are very much effective even at nanogram per ml

concentration, that means they work even at very low expressions, at this low

concentrations the others are failed to do so(Prof Oktem; Advances in TransGenic Plant

utilization in Agriculture). A stable expression of transgenic maiz line was reported by

Estrunch J.J..1998. A transgenic cotton variety [cot102] expressing a vip3A gene also

reported recently (Environmental protection agency.2003)

METHODO[OGY AND WORKP[AN:

1st year:

[1] Identification of target gene sequence: relevant VIP3 gene sequence will be identified by

using Gene Bank Database.

[2] Alteration of Gene sequence: addition of plant starting codon sequence and terminator

sequences in such a way that, the native three dimensional structure of active site remains

same/functional.

[3] Synthetic preparation of gene and transformed into suitable bacterial host for

expressional analysis.
2nd year:

[1] Identification ofa gene sequence: By using Gene Bank Database.

[2] Designing of specific primers fora gene.

[3] Isolation of a gene by PCR.

3re year:

[1] Vector construction: A suitable plant expression vector containing promoter, selectable

marker, Gene of interest and Termination sequence will be constructed.

[2] Transformation into selected crop

[3] Selection and Regeneration of Transformants.

4th year:

[1] Confirmation of transformants by PCR and Southern Bloting technique.