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marker.
INTODUCTION:
Year by year population of the world is growing dramatically, for instance in 1960,s Indian
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
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
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
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
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
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.
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
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
9.Y safety concern: (Syngenta seeds Inc, USA) The protein, gene is vigorously tested
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
genes(From aa) particularly . These two genes are most
coleopteran species. These two genes are patented by Monsanto Company, USA.
As far as coming to Indian context only one GM crop that iscotton 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
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
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 ofresistance 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
Estrunch J.J..1998. A transgenic cotton variety [cot102] expressing a vip3A gene also
1st year:
[1] Identification of target gene sequence: relevant VIP3 gene sequence will be identified by
[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:
3re year:
[1] Vector construction: A suitable plant expression vector containing promoter, selectable
4th year: