TECHNICAL FIELD

The present disclosure is in the area of agricultural biotechnology, wherein two transgenic cotton events, viz., MLS9124 and MLS9878 are disclosed. The present disclosure relates specifically to an expression cassette comprising a nucleotide sequence coding for CrylC gene in plants of genus Gossypium. The disclosure also provides vectors and host cells tansformed by the said expression cassette and corresponding methods thereof. The disclosure also comprises method of codon optimizing the said gene, and conferring of resistance to plants of genus Gossypium from important pests or insects of cultivated crops causing extensive economic damage.

BACKGROUND AND PRIOR ART OF THE DISCLOSURE

Pests cause extensive economic damage of crop plants worldwide and necessitate extensive use of chemical pesticides for control. The losses due to damages by insects cause significant losses in crop yields, a situation that the burgeoning population worldwide can ill-afford. The consequent fallout of this extensive pesticide usage is the damage to the ecosystem caused by leaching into the soils as well as by run-off into the water bodies, thus disturbing the flora and fauna. Pesticides, being generally non-preferential for the target organisms, also affect beneficial insects. The residual pesticides in the crops get into the food chain causing severe damages to the living system, including humans where instances of physical deformities have been reported.

Spodoptera litura, a pest of cotton and tobacco, is also known as the Oriental leafworm moth, Cluster caterpillar, Cotton leafworm, Tobacco cutworm, Tropical armyworm, Taro caterpillar, Tobacco budworm, Rice cutworm, and Cotton Cutworm. This moth is found in Asia, with some specific problematic pest population reports occurring in Cambodia, Hong Kong, India, the Pacific islands, Guam, American Samoa, and Hawaii. In Australia, it is found in northern two thirds of the country. It is established in the U.S., and it is a pest of national, regulatory concern. Besides cotton and tobacco, this insect causes extensive damage in many vegetables and is a major pest in corn as well as a minor pest in rice.

One of the most widely used Bacillus thuringiensis strains used in the biological control of insect pests of crops is aizawai which carries the crystal protein CrylC.

The very high insecticidal activity of this Cry protein on the diamond-back moth and the army worm (Plutella xylostella, and Spodoptera spp. respectively) combined with the impeccable safety record of the strains carrying this crystal protein has been primarily responsible for this widespread use. Extensive studies on the biosafety of this strain have been done, mostly in the US and details have been published under the joint sponsorship of the UNEP, ILO, WHO and the International Program of Chemical Safety (1).

The US Environmental Protection Agency (EPA) has approved the registration of several formulations, either Bt strains or Pseudomonas fluorescens engineered to express CrylC protein as safe pesticides to be used in all agricultural crops including fruits, vegetables, corn, cotton and nuts; turf; forests; ornamentals; landscape trees; nursery crops. The long history of safe use of these formulations even in crops which are eaten raw like lettuce attest to the high degree of safety of the organism and the CrylC protein that is expressed in them. Many commercial products by some of the leading companies in the world like Abbot (Xentari, Florbac) and Novartis (Certan) contain the expressed CrylC protein and are in wide-spread use in many countries (2).

The gene coding for the CrylC family of proteins has been extensively studied. A search of the GENBANK database shows many sequences of this family of protein deposited. A non-exhaustive list is presented in table 1. In a very significant recent study (3), CrylC expressing broccoli plants were exposed to the laboratory bred resistant strains of diamond back moth, Plutella xylostella in order to understand the effect of the CrylC protein on the parasitoid, Diadegma insulare, which is a natural enemy of Plutella. The results of this study provide the first clear evidence of the lack of hazard to a parasitoid by a Bt plant, compared to traditional insecticides.

An alternative to the use of biological control has been the implementation of transgenic technology. Crop species have been genetically modified to express different Cry genes to control insects. The earliest commercial disclosure of this technology has been Bollgard that expresses the Cry 1 Ac gene in cotton and this was developed by Monsanto (10). Bollgard was developed to resist the bollworm Helicoverpa armigera and commercial cultivation was practised in the United States in 1996. Subsequently, Bollgard II was developed by Monsanto that had the stacked combination of Cry 1 Ac and Cry2Ab and was put into commercial cultivation in 2000 to delay the onset of resistance to the Bt toxin by the pest (10, 11).

Studies on effects of two transgenic cotton lines carrying a CrylA gene on Spodoptera litura, were conducted during 2002-2005 in the cotton planting region of the Yangtze River valley of China. Results showed that the Spodoptera larvae had low susceptibility to Bt cotton (12). There was no significant difference in larval population densities in conventional and Bt cotton fields. However, the larval populations of the insect on conventional plants treated with chemical insecticides for control of target pest of Bt cotton were significantly lower than that in Bt cotton fields. These results indicated that Spodoptera litura had the potential to become a major and alarming pest in Bt cotton fields, and therefore efforts to develop an effective alternative management strategy was needed. The efficacy of CrylC in controlling insects that have developed resistance to CrylA toxins in cauliflower is reported (13).

CrylC was expressed in corn to harbour resistance to Spodoptera frugiperda (15). This was not a codon-optimized gene. Insect resistant transgenic indica rice has been reported where CrylC was expressed (16). Several patents exist for CrylC but the genes protected in these code for the protein found in Bacillus thuringiensis subspecies aizawai (17-26).

STATEMENT OF THE DISCLOSURE

Accordingly, the present disclosure relates to a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l; an expression cassette set forth as SEQ ID No.3, comprising CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3' untranslated region; a vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed above; a transformed host cell comprising the vector as claimed above; a method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of: (a) inserting an expression cassette set forth as SEQ ID No.3 into a vector, and (b) transforming a host cell with said vector to obtain the transformed host cell; a transgenic cell comprising an expression cassette as claimed above; a method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of: (a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No.l, (b) inserting an expression cassette as claimed above into a vector and transforming a host cell with said vector to obtain a transformed host cell, and (c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium; a transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l in a plant member of genus Gossypium or any part thereof; a method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of: (a) extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left border region of the plant DNA and right border region of the plant DNA or any combination thereof, and (b) detecting and analyzing the amplicon to detect the presence of said transgene in the transgenic plant; primers set forth as SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No. 19; and a method of codon optimizing CrylC gene in plant member of genus Gossypium, said method comprising acts of: (a) preparing a codon usage table, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton, and (b) using said table for codon optimizing the CrylC gene.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES

In order that the disclosure may be readily understood and put into practical effect,
reference will now be made to exemplary embodiments as illustrated with reference to
the accompanying figures. The figure together with a detailed description below, are
incorporated in and form part of the specification, and serve to further illustrate the
embodiments and explain various principles and advantages, in accordance with the
present disclosure where

Figure 1 illustrates pairwise alignment of the codon optimized CrylCGh gene with
the 1st 1890 bp of the CrylC gene (4).

Figure 2 illustrates a map of the CrylCGh expression cassette in the transgenic
cotton event MLS9124 and MLS9878.

Figure 3 illustrates a map of the binary vector pMH72.

Figure 4 illustrates a map of the plasmid pMH82 with T -DNA containing two Cry
1C expression cassettes repeated in tandem used for the generation of the cotton
transgenic events MLS9124 and MLS9878.

Figure 5 illustrates southern hybridization of event MLS9124 digested with EcoRI
and HindIII.

Figure 6 illustrates southern hybridization of event MLS9878 digested with EcoRI
and HindIII.

Figure 7 illustrates a diagnostic PCR process for the amplification of the left border
junction of transgenic cotton event MLS9878.

Figure 8 illustrates a diagnostic PCR process for the amplification of the right border
junction of transgenic cotton event MLS9878.

Figure 9 illustrates a diagnostic PCR to locate the unique intermediate region
between the two expression cassettes in MLS9124 and MLS9878.

Figure 10 illustrates a comparative efficacy of CrylC in transgenic cotton events
MLS9124 and MLS9878 in Spodoptera litura (A) and Helicoverpa armigera (B).

Figure 11 illustrates a diagnostic PCR process for the amplification of the right border junction of transgenic cotton event MLS9124.

DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure relates to a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l.

The present disclosure further relates to an expression cassette set forth as SEQ ID No.3, comprising CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3' untranslated region.

In an embodiment of the present disclosure, the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

The present disclosure further relates to a vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed above.

In an embodiment of the present disclosure, the vector is selected from a group comprising an expression vector, replicable vector, transformation vector, binary vector and shuttle vectors or any combination thereof.

In another embodiment of the present disclosure, the vector is preferably binary vector and wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

The present disclosure further relates to a transformed host cell comprising the vector as claimed above.

The present disclosure further relates to a method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of: (a) inserting an expression cassette set forth as SEQ ID No.3 into a vector, and (b) transforming a host cell with said vector to obtain the transformed host cell.

In an embodiment of the present disclosure, the expression cassette comprise CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3' untranslated region.

In another embodiment of the present disclosure, the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for Cry 1C protein.

In yet another embodiment of the present disclosure, the host cell is Agrobacterium.

In still another embodiment of the present disclosure, the transforming of the host cell is carried out using techniques selected from a group comprising electroporation, tri-parental mating, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer and or any combination thereof.

The present disclosure further relates to a transgenic cell comprising an expression cassette as claimed above.

In an embodiment of the present disclosure, the transgenic cell is a transgenic plant cell exhibiting resistance to lepidopteran insect(s) or pest(s) infestation; and wherein the plant is a member of genus Gossypium.

The present disclosure further relates to a method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of: (a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No.l, (b) inserting an expression cassette as claimed above into a vector and transforming a host cell with said vector to obtain a transformed host cell, and (c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium.

In an embodiment of the present disclosure, the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

In another embodiment of the present disclosure, the host cell is Agrobacterium.

In yet another embodiment of the present disclosure, transforming the host cell is carried out using techniques selected from a group comprising electroporation, tri-parental mating, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method and liposome mediated transfer or any combination thereof.

In still another embodiment of the present disclosure, the infection and tissue culturing comprises incubating cotton leaf with the transformed host cell; transferring and incubating the cotton leaf in bacterial selection medium followed by incubation in plant selection medium; transferring to embryogenesis medium till the occurance of embryogenesis followed by sub-culturing; transferring to suspension medium followed by germination medium; transfering to basal medium after the growth of leaves and root hairs; hardening followed by transfer to soil and greenhouse to obtain the said transgenic plant.

The present disclosure further relates to a transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. 1 in a plant member of genus Gossypium or any part thereof.

The present disclosure further relates to a method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of: (a) extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left border region of the plant DNA and right border region of the plant DNA or any combination thereof, and (b) detecting and analyzing the amplicon to detect the presence of said transgene in the transgenic plant.

In an embodiment of the present disclosure, the primers are selected from a group comprising SEQ ID No. 11 corresponding to the left border region of the transgene ,
SEQ ID No. 13 corresponding to the right border region of the transgene, SEQ ID No. 12 correspdonding to the left border region of the plant DNA and SEQ ID No. 14 corresponding to the right border region of the plant DNA or any combination thereof.

The present disclosure further relates to primers set forth as SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No. 19.

In an embodiment of the present disclosure, the primers having SEQ ID Nos. 11, 13, 15 and 19 are sense primers and the primers having SEQ ID Nos. 12, 14, 16 and 18 are anti-sense primers.

The present disclosure further relates to a method of codon optimizing CrylC gene in plant member of genus Gossypium, said method comprising acts of: (a) preparing a codon usage table, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton, and (b) using said table for codon optimizing the CrylC gene.

In an embodiment of the present disclosure, the nucleotide sequence is codon optimized for plants of genus Gossypium.

A codon optimized gene is necessary to code for the CrylC protein that would confer resistance to 5". litura in cotton, tobacco, tomato, cabbage, cauliflower, broccoli, rice and maize. Another object of the disclosure is to incorporate it in a stack with other Bt genes to delay the onset of breakdown of resistance to pests. CrylC is also responsible to confer resistance to the crop plants to other lepidopteran pests.

In an embodiment of the present disclosure, the cotton plant is selected from a group comprising Gossypium hirsutum, Gossypium barbadense, Gossypium arboretum and Gossypium herbaceum. Preferably, the cotton plant is Gossypium hirsutum.

CrylC is a potent insecticidal protein found in several subspecies of B. thuringiensis that is effective against a wide range of lepidopteran pests of crops. The range of the crops is very diverse and includes, but is not restricted to, cotton, tobacco, tomato, cauliflower, broccoli, rice and maize. Besides conferring resistance to lepidopteran pests that are not affected by the CrylA series of insecticidal proteins, this gene, when stacked with any CrylA protein also helps the delay of onset of resistance to CrylA group of proteins expressed in transgenic crops in the target pests.

This disclosure relates to two transgenic cotton events that carry a unique CrylC protein gene that is optimized based on the codon usage for the most constitutively expressed proteins in cotton. This gene, when expressed in tobacco and cotton, demonstrates efficacy against major insect pests hitherto uncontrolled by any CrylA protein.

The N-terminal 630 amino acid sequence of said CrylC gene was codon optimized using a codon usage table for highly constitutively expressed genes based on the codon preference for cotton (27).
The Pairwise alignment of the codon optimized CrylCGh gene with the 1st 1890 bp of the CrylC gene is provided in figure 1.

Based on the codon preference for cotton (27), a table was created for the most constitutively expressed genes (Table 2):

The designed sequence of the gene is given below as SEQ ID No. 1. The gene was designed using the N-terminal 630 amino acids [SEQ ID No. 2] of the CrylC protein from Bacillus thuringiensis subspecies entomocidus (4, 30), unlike the sequences cited in references 17-26, which are for B. thuringiensis subspecies aizawai. Factors that were taken into consideration while designing the gene were GC content, TA doublet avoidance in the second and third positions of codons, transcription termination signal (AGTNNAA), splicing signals (5' splice junction: CRG/GTAAGT; 3' junction: YNAG/N) and polyadenylation signals (AATAAA, AATGAA, AATAAT, GATAAA, AATAAG, AATATT).

The gene thus obtained was cloned in a binary vector pMH72 (figure 3, SEQ ID No. 4) which was developed to express the nptll gene for plant selection from pGA643 (28) in pPZP201 (29). The gene was cloned to be driven by the cauliflower mosaic virus (CaMV) double 35S promoter (D35SP) and the 35S 3' UTR was used for transcriptional termination. The resultant binary plasmid pMH82 as described in figure 4 and its T-DNA sequence [given in SEQ ID No. 5], was mobilized into Agrobacterium tumefaciens EHA105. The mobilization of pMH82 into Agrobacterium tumefaciens EHA105 can be carried out using various conventionally known techniques of gene transfer which include microinjection, electroporation, tri-parental mating, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer. In an embodiment of the present disclosure, electroporation method is used eherein, electrocompetent Agrobacterium tumefaciens cells are prepared and frozen in glycerol at -70°C. The cells are thawed on ice prior to electroporation. Approximately, 50 ng of plasmid DNA is mixed with the electrocompetent cells and transferred to a pre-chilled cuvette. A pulse of 1.8 kV/zero millisecond is applied (feature Eel on BioRad MicroPulser Electroporator). The cells are recovered in 1 ml of Luria Bertani Medium for 45 minutes by shaking at 200 rpm at 26°C. The recovered culture is further diluted 25 fold and plated on a minimal medium plated with kanamycin. This plate is incubated at 26°C for 2 days. The transformed cells are selected and used further.

Hypocotyl bits of cotton variety Coker are infected with the Agrobacterium cultures harbouring theplasmid pMH82. Embryogenic calli are obtained and regenerated following which regenerants are assayed for resistance to 5". litura, Earias vitella and Helicoverpa armigera.

The uniqueness of the construct in pMH82 arises from 2 aspects, viz., the presence of dual expression cassettes [each expression cassette set forth as SEQ ID No. 3] and a unique 57 bp region in between the two expression cassettes, as given in SEQ ID No. 6.
Map of the CrylCGh expression cassette in the transgenic cotton event MLS9124 and MLS9878 is provided in figure 2.
A Flow chart depicting the course of events for obtaining transgenic cotton plants is provided as below:

Hypocotyl segments of cotton or tobacco leaf discs

Incubate with Agrobacterium culture in dark (36-48 hours)

Wash with sterile water
1 Transfer to bacterial selection medium, keep in dark for 5 days

Transfer to plant selection medium, changing medium periodically every 15 days.
Carry this out for at least 3 passages.

Transfer to an embryogenesis medium till embryogenesis occurs, sub culturing every
4-6 weeks

After embryogenesis transfer to suspension medium
Transfer slightly elongated embryos to a charcoal medium
Transfer to germination medium in vermiculite first in diffused light and then to light
When true leaves and root hairs appear transfer to a basal medium
Harden the plants in coco peat cum perlite
Transfer to soil and into the greenhouse

Two independent transgenic cotton events, viz., MLS9124 and MLS9878, were further studied by Southern hybridization (31), analysis of the site of integration using TAIL-PCR (32), ELISA for studying levels of protein expression and bioassays by challenging the events with Spodoptera litura.

For Southern hybridization, 20 ug genomic DNA was digested with either Hindlll or EcoRI overnight and separated on a 0.8% agarose gel in TBE. The gel was transferred to Nylon membrane by downward capillary transfer and hybridized to either a 2.317 kb nptll probe [SEQ ID No. 7] or a 1.954 kb CrylCGh probe[SEQ ID No. 8]. The probes were labeled with a-32P dCTP using a randomly primed extension with Klenow and hybridized overnight with formamide. Washed gels were exposed to Kodak X-OMAT films.

Southern Hybridization

Southern hybridization of event MLS9124 digested with EcoRI and Hindlll (figure 5);
comprises:

(a) Hybridization with probe I (Nptll probe: EcoRI-Hindlll fragment from
pMH82-2317bp)

(b) Hybridization with probe II (CrylCGh probe: Xhol fragment from pMH82-
1954 bp)

Southern hybridization of event MLS9878 digested with EcoRI and Hindlll (Figure 6); comprises:

(a) Hybridization with probe I (Nptll probe: EcoRI-Hindlll fragment from
pMH82-2317bp)

(b) Hybridization with probe II (CrylCGh probe: Xhol fragment from pMH82-
1954 bp)

HindIII are given between the two panels [event MLS9878]

The results are as illustrated in Figure 6, wherein the legends for both panels are provided as below:

The two transgenic events were further analyzed by PCR for the presence of the unique region as described in SEQ ID No. 6 between the 2 expression cassettes. The primer sequences are described in SEQ ID No. 15 (forward primer) and SEQ ID No. 16 (reverse primer). The expected amplicon size was 975 bp.

Diagrammatic representation of diagnostic PCR process for the amplification of the left and right border junction of the transferred DNA [T-DNA] within the transgenic cotton events MLS9878 are illustrated in figures 7 and 8 respectively.

Transgenic cotton events not harbouring the T-DNA of the plasmid pMH82 were also subjected to this PCR and these samples did not amplify.

The results are as illustrated in Figure 9, wherein the legend for the gel loaded with different samples is provided as below:

The 975 bp amplicon from both the events MLS9124 and MLS9878 is sequenced and is set forth as SEQ ID NO. 17 which shows the presence of the unique 57 bp region described in SEQ ID No. 6.

Junction Analysis

In an embodiment of the present disclosure, junction analysis studies for both the
transgenic cotton events are carried out at a lab scale.

The left and right border junction sequence of the transgenic cotton event MLS9878 are set forth as SEQ ID Nos. 9 and 10, respectively.

T-DNA specific primer amplifying the left border junction sequence of the transgenic cotton events MLS9878 is set forth as SEQ ID No. 11, whereas the T-DNA specific primer amplifying the right border junction sequence of the transgenic cotton events MLS9878 is set forth as SEQ ID No. 13.

The corresponding cotton genome specific primer amplifying the left border junction sequence of the transgenic cotton event MLS9878 is set forth as SEQ ID No. 12, whereas the cotton genome specific primer amplifying the right border junction sequence of the transgenic cotton event MLS9878 is set forth as SEQ ID No. 14.

The right border junction sequence of the transgenic cotton event MLS 9124 is set forth as SEQ ID No. 18. The corresponding T-DNA specific primer amplifying the right border junction sequence of the transgenic cotton events MLS9124 is set forth as SEQ ID No. 19 and the cotton genome specific primer amplifying the right border junction sequence of the transgenic cotton event MLS9124 is set forth as SEQ ID No. 20.

Diagrammatic representation of diagnostic PCR process for the amplification of the right border junction of transgenic cotton events MLS9124 is illustrated in figure 11, wherein the legend for the gel loaded with different samples is provided as below:

Spodoptera litura and Helicoverya armigera Bioassays:

Transgenic cotton events MLS9124 and MLS9878 were assayed for resistance to S. litura and Helicoverpa armigera up to the T4 generation and mortality up to 100% was observed to be carried through generations.

First fully expanded terminal leaves from unsprayed plots expressing various transgenes are bioassayed for bioactivity against Helicoverpa armigera and Spodoptera litura larvae. These bioassays were carried out at time intervals of 64, 78, 92, 106, 120 and 134 days after planting, in Non transgenic event (control), Event MLS9124 and Event MLS9878.
Length of the sampled leaves is measured approximately 5 cm across and is removed with as much petiole attached as possible. Leaves from each replicate are kept separate in labeled paper sacks and placed in plastic cooler containing cooling packs for transport to the laboratory.

Individual leaves are placed in a Petri dish having wet whatman filter paper and infested with a single day old fed larva (10 dishes/plot) for a total of 40 larvae/trait. Bioassays are kept at a constant temperature (25-30°C and relative humidity greater than 50 %) prior to assessment. After five days after exposure (DAE), larvae are prodded with a camel-hair brush and considered alive if coordinated movement is observed. Larvae from each replicate are scored according to the criteria in Table 8:

Percent survival and development of larvae is analyzed using analysis of variance (ANOVA). Insect bioassay results of the two transgenic cotton events MLS9124 and MLS9878 for efficacy against Spodoptera litura are summarized in Figure 10 (A) and
10 (B). The data is captured at different stages of the growth cycle of the transgenic
cotton events. In an embodiment of the present disclosure, the bar diagram for each
event per insect has one bar for the average worm size. This is calculated as follows:
Each category of survival score is assigned a factor which is designated as-

Ll-0.5
L2-1
L3-2

Further, (number of LI x 0.5) + (number of L2 x 1) + (number of L3 x 2) = total worm size.

Total worm size / Total number of survived larvae = Average worm size

In an embodiment, the total number of survived larvae is 40, wherein LI is 20,
L2 is 11 and L3 is 9.

Therefore the total worm size is (20 X 0.5)+(l 1X1)+(9X2) = 39 and the average
worm size is-Total worm size /Total number of survived larvae= 39/40 = 0.975.

In an exemplary embodiment, the average worm size for constructing a bar diagram is calculated as a percentage of the L3 factor, i.e., 2. Therefore, if the value of average worm size is found to be 0.975, it is represented in the bar diagram as (0.975/2) x 100 = 48.75%.

Earias vitella Bioassays:

Squares (flower buds) of approximately 4-6 mm thickness from unsprayed plots were bioassayed for activity against Earias vittella larvae.

Sampled squares are removed with as much petiole attached as possible. Squares from each replicate are kept separate in labeled paper sacks and placed in plastic cooler containing cooling packs for transport to the laboratory. Individual squares are placed into a Petri dish having wet whatman filter paper and infested with a two day old fed larva for a total of 40 larvae/trait. Bioassays are kept at a constant temperature (25-30°C and relative humidity greater than 50%) prior to assessment. After five days after exposure (DAE), larvae are prodded with a camel-hair brush and considered alive if coordinated movement is observed.

Insect bioassay results of the two transgenic cotton events MLS9124 and MLS9878 for efficacy against Earias vitella are summarized in Tables 9 and 10 respectively.


Protein expression data:

The expression of the CrylC protein as estimated by quantitative ELISA was in the
range 0.41 to 4.67 ug/gm fresh weight of leaf. This is explained in Table 11 below.

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We Claim:

1. A nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l.

2. An expression cassette set forth as SEQ ID No.3, comprising CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3' untranslated region.

3. The nucleotide sequence as claimed in claim 1 and the expression cassette as claimed in claim 2, wherein the nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

4. A vector comprising sequence set forth as SEQ ID No. 5, having an expression cassette as claimed in claim 2.

5. The vector as claimed in claim 4, wherein the vector is selected from a group comprising an expression vector, replicable vector, transformation vector, binary vector and shuttle vectors or any combination thereof.

6. The vector as claimed in claims 4 and 5, wherein the vector is preferably binary vector and wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

7. A transformed host cell comprising the vector as claimed in claim 4.

8. A method of obtaining a transformed host cell comprising an expression cassette set forth as SEQ ID No.3, said method comprising acts of:

a) inserting an expression cassette set forth as SEQ ID No.3 into a vector; and
b) transforming a host cell with said vector to obtain the transformed host cell.

9. The method as claimed in claim 8, wherein the expression cassette comprise
CaMV 35S promoter, nucleotide sequence set forth as SEQ ID No. 1 or a
nucleotide sequence comprising sequence set forth as SEQ ID No.l and 35S 3'
untranslated region.

10. The method as claimed in claim 9, wherein said nucleotide sequence codes for CrylC gene and corresponding amino acid sequence set forth as SEQ ID No. 2 codes for CrylC protein.

11. The method as claimed in claim 8, wherein the host cell is Agrobacterium.

12. The method as claimed in claim 8, wherein the transforming of the host cell is carried out using techniques selected from a group comprising electroporation, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method, liposome mediated transfer and tri-parental mating or any combination thereof.

13. A transgenic cell comprising an expression cassette as claimed in claim 2.

14. The transgenic cell as claimed in claim 13, wherein the transgenic cell is a transgenic plant cell exhibiting resistance to lepidopteran insect(s) or pest(s) infestation; and wherein the plant is a member of genus Gossypium.

15. A method of obtaining a transgenic plant member of genus Gossypium, said method comprising acts of:

a) codon optimizing nucleotide sequence of native CrylC gene to obtain nucleotide sequence set forth as SEQ ID No.l;
b) inserting an expression cassette as claimed in claim 2 into a vector and transforming a host cell with said vector to obtain a transformed host cell; and
c) infecting the plant with the transformed host cell followed by tissue culturing to obtain a transgenic plant member of genus Gossypium.

16. The method as claimed in claim 15(b), wherein the expression cassette is either a single copy sequence or tandem repeat sequence separated by an intervening sequence set forth as SEQ ID No.6.

17. The method as claimed in claim 15, wherein the host cell is Agrobacterium.

18. The method as claimed in claim 15, wherein the transforming the host cell is carried out using techniques selected from a group comprising electroporation, microinjection, genegun method, PEG mediated transfer, Calcium phosphate method and liposome mediated transfer or any combination thereof.

19. The method as claimed in claim 15 (c), wherein the infection and tissue culturing comprises incubating cotton leaf with the transformed host cell; transferring and incubating the cotton leaf in bacterial selection medium followed by incubation in plant selection medium; transferring to embryogenesis medium till the occurance of embryogenesis followed by sub-culturing; transferring to suspension medium followed by germination medium; transfering to basal medium after the growth of leaves and root hairs; hardening followed by transfer to soil and greenhouse to obtain the said transgenic plant.

20. A transgenic transformation event MLS9124 or MLS9878, said event comprising a nucleotide sequence set forth as SEQ ID No. 1 or a nucleotide sequence comprising sequence set forth as SEQ ID No. 1 in a plant member of genus Gossypium or any part thereof.

21. A method of detecting presence of a trangene comprising an expression cassette set forth as SEQ ID No.3, in a transgenic plant member of genus Gossypium, said method comprising acts of:

a. extracting DNA from the plant source and performing nucleic acid amplification of junction regions of the transgene and the plant to obtain an amplicon, said amplification carried out by primers corresponding to the regions selected from a group comprising left border region of the transgene, right border region of the transgene, left
border region of the plant DNA and right border region of the plant DNA or any combination thereof;

b. detecting and analyzing the amplicon to detect the presence of said
transgene in the transgenic plant.

22. The method as claimed in claim 21, wherein the primers are selected from a group comprising SEQ ID No. 11 corresponding to the left border region of the transgene , SEQ ID No. 13 corresponding to the right border region of the transgene, SEQ ID No. 12 correspdonding to the left border region of the plant DNA and SEQ ID No. 14 corresponding to the right border region of the plant DNA or any combination thereof.

23. Primers set forth as SEQ ID No. 11, SEQ ID No. 12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No. 15, SEQ ID No. 16, SEQ ID No. 18 and SEQ ID No. 19.

24. The primers as claimed in claim 23, wherein the primers having SEQ ID Nos. 11, 13, 15 and 19 are sense primers and the primers having SEQ ID Nos. 12, 14, 16 and 18 are anti-sense primers.

25. A method of codon optimizing CrylC gene in plant member of genus Gossypium, said method comprising acts of:

a) preparing a codon usage table as described in Table 2, for plurality of constitutively expressed genes, on the basis of GC content, TA doublet avoidance in second and third positions of codons, transcription termination signals, splicing signals and polyadenylation signals, from conventionally known constitutively expressed genes in cotton; and

b) using said table for codon optimizing the CrylC gene.

26. The nucleotide sequence as claimed in claim 1, the expression cassette as claimed in claim 2 and the method as claimed in claim 9, wherein the nucleotide sequence is codon optimized for plants of genus Gossypium.