Genetic Transformation of Cotton for Bollworm Helicoverpa armigera (Hubner) Resistance using Chimeric cry2AX1 Gene

Cotton (


Introduction
Cotton is the leading fiber crop produced in the world and the most important crop in India.India is the largest producer of cotton in the world, the second largest exporter of raw cotton (www.cottoninc.com).The growth and productivity of cotton crop is often hampered by various biotic as well as abiotic stress factors which results in yield reduction and poor quality of cotton fibres.One of the major limiting factors, which affect cotton production in India, is the incidence of pests, especially bollworms, causing more than 50 per cent yield loss 1 .Helicoverpa armigera is the most common bollworm of cotton in India and can result in yield loss ranging from 30-80% 17 .
The limited genetic variability for bollworm resistance in cotton germplasms makes the task of developing bollworm-resistant genotypes very difficult 15 .However, developing improved cotton cultivars/hybrids with desirable traits by conventional cross breeding methods is time consuming and laborious.Under these circumstances traits from other organism could be horizontally transferred by use of genetic engineering techniques.Genetic engineering offers successful production of transgenic plant with desirable traits within stipulated period of time.The use of proteins encoded by cry genes of Bacillus thuringiensis (Bt) for insect pest management has emerged as a potent tool, being pesticide free, environment friendly and highly specific against target insects due to presence of specific receptor and non toxic to majority of beneficial insects and vertebrates, due to lack of specific receptor 6,27 .Genetically modified (GM) crops expressing Bt toxin for imparting resistance against different insect pests have been commercially cultivated in several countries.Socio-economic benefit of GM crops has been well proven through meta-analysis studies on impact of GM crops.Adoption of GM technology has reduced chemical pesticide use by 37%, increased crop yields by 22%, and increased farmer profits by 68% 16 .Agrobacterium-mediated gene transfer method has been used successfully for transformation of numerous dicot species.Cotton engineered with different insecticidal protein gene(s) from Bt has been reported by many authors to provide protection to the plants against different lepidopteran insect pests 18,30,31,35,36 .Owing to the difference in structure and insecticidal mechanism, cry2A genes are promising candidates for the management of insects in crop plants 12 .The chimeric Cry2AX1 protein was found to be more toxic than its parental proteins (Cry2Aa and Cry2Ac) 34 .In the light of the above developments, the present study was conducted to produce transgenic cotton plants expressing cry2AX1 gene, using Agrobacteriummediated transformation and to evaluate the resistance of the developed plants to H. armigera.

Binary Vectors and Strain
The pC2300-En35S-2AX1 construct containing cry2AX1 gene 14 (Figure 1 (a)) and pC2300-En35S-ctp2AX1 25 containing cry2AX1 with chloroplast transit peptide sequence (Figure 1 (b)) (both were driven by EnCaMV35S promoter and nos-polyA in pCAMBIA 2300 backbone 9 , were mobilized into Agrobacterium strain LBA4404. Both these vectors harbor the neomycin phosphotranferase (nptII) gene (driven by CaMV35S promoter and polyA), which confers resistance to kanamycin used for plant selection.Agrobacterium strain LBA4404 har bouring these binary vectors was used for transformation of cotton; grown on YEP [1% Yeast extract, 1% Peptone and 0.5% NaCl pH 7.0] media containing kanamycin 50 μg/ml and rifampicin 20 μg/ml in an incubator at 28°C and utilized for transformation of embryogenic cotton calli.

Agrobacterium Mediated Transformation of Cotton
Sulphuric acid delinted seeds of Coker 310 were surface sterilized with 70% ethanol for 1 min and washed thrice with sterile distilled water.They were again surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 10 min, followed by three washes with sterile distilled water and the seeds were blot dried on sterile tissue paper.The sterile seeds were germinated on a half strength MS basal medium 23 in germination bottle, supplemented with 15 g/L sucrose and 8 g/L agar.The germination bottles were incubated for one week in the culture room at 25 ± 2°C under 16/8 hr photoperiod Cotyledon (~10 mm 2 ) and hypo-cotyledon (8 mm long) sections were excised from seven day old cotton seedlings and inoculated in callus induction medium (CIM) supplemented with 0.1 mg/L 2, 4-D and 0.5 mg/L kinetin, 30 g/L maltose solidified with 3.6 g/L phytagel for callus induction.The explants were sub-cultured in CIM after 2 weeks.After one month, induced fresh calli were separated from the explants and sub-cultured in the same medium for another one month for calli proliferation.Highly proliferating calli of two to three months old were transferred to MS basal medium and maintained for two month on LB: left border, polyA: CaMV35S, nptII: neomycin phosphotransferase gene, CaMV35S2: Cauliflower mosaic virus 35S duplicated promoter, EnCaMV35S: Enhanced cauliflower mosaic virus 35S promoter, ctp: chloroplast transit peptide, cry2AX1: gene of interest, Tnos: nopaline synthase terminator; RB: right border upward capillary transfer protocol.The transferred DNA was cross linked by UV exposure at 1200 μJ/min for 1 min.For hybridization, an 800 bp internal region of cry2AX1 gene was used as a probe.Probe DNA was labeled with α 32 P deoxycytidine triphosphate (dCTP) using the decalabel DNA labeling kit (Thermo Scientific Inc, Waltham, USA) and added into hybridization solution.Prehybridization was carried out for 1h and hybridization for 16 h at 60⁰C.After hybridization, the blot was washed with 2X SSC (saline sodium citrate) + 0.1% SDS (sodium dodecyl sulphate) for 15 min, followed by 10 min in 0.5X SSC + 0.1% SDS and final wash with 0.1X SSC+ 0.1% SDS for 5 minute.All washings were carried out at 60⁰C and the blot was exposed to X-ray film.

ELISA Analysis
A double-antibody sandwich Enzyme Linked Immuno Sorbent Assay (ELISA) was used to detect the presence of the Cry2AX1 protein expressed in the leaves of transgenic cotton plants.Experiments were performed with double sandwich quantitative Cry2A ELISA kit (Envirologix, Portland, USA).Proteins from leaf samples of transformed and untransformed cotton plants were extracted using the protein extraction buffer provided in the kit.Each sample was replicated twice.Leaf extract was diluted to fit in the linear range of the provided Cry2A standards and steps were performed according to manufacturer's instructions.Optical density (O.D.) was read at 450 nanometres in an ELISA plate reader (Biotek, USA).The quantity of Cry2AX1 protein present in the sample was calculated by referring to standard graph generated with Cry2A calibration standards and represented in µg/g fresh weight of leaves.

Insect Bioassay
Detached leaf bit bioassay was carried out to determine the degree of insect resistance in ELISA positive T 0 transgenic cotton plants under laboratory condition with H. armigera neonates.Leaf bit from T 0 transgenic and control cotton plants were placed in a wet filter paper in Petri plates and used for bioassay.One neonate larva of H. armigera was released on each leaf bit.Each treatment was replicated thrice and for each replication ten larvae were taken.The experiment was carried out at 27±1ºC with about 65 per cent relative humidity.Larval mortality was recorded after 48 hours at 24 hours interval for seven days. the same medium to achieve callus maturation.After callus maturation (total age of callus-120 days) good proliferating callus (loose, friable and cream-coloured) were transferred to MS medium supplemented with 1.9 g/L KNO 3 , 30 g/L maltose solidified with 3.6 g/L phytagel [referred to as somatic embryogenesis medium (SEM)] for the induction of embryogenic callus.After one month of culture, vigorously growing, friable, loose and white embryogenic calli were used for co-cultivation.The cultures were maintained at 26⁰C under a 16/8-h photoperiod (white fluorescent bulb) at a light intensity of 100 μmoles m -2 sec -1 in a growth chamber (Sanyo Electric Co Ltd, Moriguchi, Japan).

PCR Analysis
DNA was isolated from putative transgenic cotton plants using CTAB method 24 .PCR screening of putative cotton transformants was performed using cry2AX1 gene specific primers (Forward 5'CCTAACATTGGTGGACTTCCAG 3' and Reverse 5' GAGAAACGAGCTCCGTTATCGT 3') and nptII gene specific primers (forward primer 5'-AGAACTCGTCAAGAAGGCGA and reverse primer 5'-CAGACAATCGGCTGCTCTGA).The PCR for the both sets of samples was carried out using a Thermal Cycler (Eppendorf, Germany) in 25 µl reaction volume containing, 2.5 µl of 10X Taq buffer, 75 μM each of dNTPs, 50 ng each of forward and reverse primers and 1.5U of Taq DNA polymerase (Bangalore Genei, India) and incubated in a thermal cycler which was programmed for 5 min preheat at 94°C and then 30 cycles of denaturation at 94°C for 1 min, annealing temperature of 58°C for 45 s and extension time of 45 s at 72°C, with a final extension at 72°C for 7 min.The plasmid DNA was used as positive control and wild type non transformed cotton plant genomic DNA sample was used as negative control for the PCR reaction.The PCR products were run on 1.0 % agarose gel and analyzed on G: BOX F3 (Syngene) gel documentation system.

Southern Blot Analysis
Ten microgram of genomic DNA (isolated from T 0 transformed and non-transformed cotton plants) was digested overnight by HindIII enzyme, electrophoretically separated on 0.8 % agarose gel using 1X TAE, denatured with 0.25 M NaOH and transferred overnight to a positively charged membrane using 20X SSC following standard integration of cry2AX1 gene in the genome of transgenic cotton plants (Figure 3 Lane 1-4).
Additional band of higher size was also observed in the transformant CETP-M1 (Figure 3 (lane 3)).The genomic DNA from non-transgenic control plant was used as a negative control and no hybridization signal was detected (Figure 3 (lane 5)), while gene specific fragments (positive control) generated hybridization signal of size ~2.1 and ~2.3 Kb, respectively as in positive control plasmids (Figure 3 (lane P1, lane P2)).

ELISA Analysis
PCR positive plants were further analyzed for the expression of Cry2AX1 protein by quantitative ELISA.Out of 11 plants tested 7 were found positive for expression of cry2AX1 and the concentration of protein ranged from 0.015 to 0.080 μg/g of fresh leaf tissue (Table 1).

Insect Bioassay
The mortality of neonates on T 0 plants ranged from 10 to 36.66 per cent, whereas the control plants showed no mortality (Table 1).Even though, the level of mortality was less, growth inhibition in surviving larvae and reduction in leaf area feeding were observed in the ELISA positive plants (Figure 4).

Plant Regeneration
A total of 11 putative transgenic plants were regenerated out of which six were generated with pC2300-En35Sctp-2AX1 construct and five with pC2300-En35S-2AX1 construct.

PCR Analysis
Eleven putative T 0 transgenic cotton plants generated were screened for the presence of cry2AX1 gene and nptII gene with specific primers targeting the coding region of cry2AX1 and nptII gene, respectively.All the eleven plants were found positive for PCR and amplified expected size of 712 bp fragment for nptII specific primers (Figure 2 (a)) and 800 bp fragments for cry2AX1 gene specific primers respectively (Figure 2 (b)).

Southern Hybridization Analysis
For southern hybridization analysis, the genomic DNA from selected four primary transformants were digested with HindIII enzyme and probed with internal fragment of cry2AX1 gene specific probe.This confirmed the   risk that field insects could develop resistance to these Bt toxins after prolonged and consistent exposure 3 .Adaptation of insects to insecticidal toxins of Bt can reduce the efficacy of toxins and thereby the benefits of the technology.We have shown that the expression of a novel synthetic Bt gene encoding the Cry2AX1 protein could be an alternative Bt gene for protection against H. armigera.PCR positive cotton transformants of cry2AX1 gene were analyzed for the level of expression of Cry2AX1 protein.Seven out of 11 PCR positive plants were found to be positive for the expression of Cry2AX1 protein, ranging from 0.015 to 0.080 μg/g fresh leaf tissue.Low and variable level of Bt protein expression in transgenic plants were reported by earlier workers.The variable level of Cry2AX1 protein observed among different transgenic lines could be envisaged for the relative location of the integrated T-DNA into the genome, so called position effects 21 ; the chromatin structure; methylation state 22 ; post-transcriptional regulation 28 and copy numbers 5,10,11 .Truncations and rearrangements have also been reported to influence the expression of the transgene 7 .The few transgenic lines which were positive by PCR but did not have expression of Cry2AX1 protein at all; this could be due to the complete inactivation of the gene because of its integration into highly repetitive DNA region of the plant's genome 28 .Integration site of transgene in the genome may have a detrimental effect on its expression.Significant differences in the level of expression of the Bt protein among the transgenic T 0 plants have been reported earlier 20,29 .
Southern blot analysis provided the additional evidence of the integration of cry2AX1 gene in cotton genome.When the DNA was digested with HindIII, the hybridization was occurred at ~2.1 and ~2.3 Kb for the transformants generated with p2300-En35S-2AX1 and p2300-EnCaMV-ctp-2AX1 constructs, respectively.The ~2.1 Kb fragment includes whole coding sequence of the cry2AX1 gene and the nos terminator whereas the ~2.3Kb fragment includes ctp-cry2AX1 fusion gene and nos terminator.This result indicated that the integrated cry2AX1 gene is intact.Besides this transformant (Lane 3) showed extra band of higher size than the expected band size, (~2.3 Kb), which indicates truncation of the cassette and subsequent integration in the cotton genome, probably due to deletion at 3' HindIII site.Similar observations of truncation/deletion of integrated T-DNA have been reported in other studies in tobacco 19,32,33 , in rice 37 and in barley 2 .No hybridization signal could be detected from the untransformed plant.

Discussion
Transgenic crop plants expressing different Bt toxin have shown significant resistance to important agricultural insect pests in addition to reduced application of pesticides and improved yield 13,26 .However, there is always a The cotton transformants showed a morality in the range 10 to 36.66 % against the neonates of H. armigera even with low level of Cry2AX1 protein.A positive correlation was established between the level of Cry2AX1 expression and per cent mortality and reduction in feeding against H. armigera larvae.Such positive correlation between expression level of Cry protein and insect mortality have been reported in many studies 4,8,20,29 .However, we could not obtain T 0 transgenic cotton event expressing higher level of Cry protein as reported earlier in transgenic tobacco, maize, rice, cotton and tomato 13,26 .

Conclusion
In the present study, we have introduced a synthetic chimeric gene, cry2AX1 into cotton to study its insecticidal activity against H. armigera.The cotton transformants showed mortality up to 36 % in neonates of H. armigera even at the lower level expression of Cry2AX1 protein.This suggests screening of a larger population of primary transformants for obtaining an event with higher expression of Bt toxin in recalcitrant plant species like cotton is necessary.

Figure 4 .
Figure 4. Detached leaf bit bioassay against H. armigera in transgenic cotton plants expressing Cry2AX1 protein.

Table 1 .
Expression of Cry2AX1 protein and mortality of H. armigera neonates in T 0 transgenic