Novel approaches to circumvent the devastating effects of pests on sugarcane

Sugarcane (Saccharum officinarum L.) is a cash crop grown commercially for its higher amounts of sucrose, stored within the mature internodes of the stem. Numerous studies have been done for the resistance development against biotic and abiotic stresses to save the sucrose yields. Quality and yield of sugarcane production is always threatened by the damages of cane borers and weeds. In current study two problems were better addressed through the genetic modification of sugarcane for provision of resistance against insects and weedicide via the expression of two modified cane borer resistant CEMB-Cry1Ac (1.8 kb), CEMB-Cry2A (1.9 kb) and one glyphosate tolerant CEMB-GTGene (1.4 kb) genes, driven by maize Ubiquitin Promoter and nos terminator. Insect Bio-toxicity assays were carried out for the assessment of Cry proteins through mortality percent of shoot borer Chilo infuscatellus at 2nd instar larvae stage. During V0, V1 and V2 generations young leaves from the transgenic sugarcane plants were collected at plant age of 20, 40, 60, 80 days and fed to the Chilo infuscatellus larvae. Up to 100% mortality of Chilo infuscatellus from 80 days old transgenic plants of V2 generation indicated that these transgenic plants were highly resistant against shoot borer and the gene expression level is sufficient to provide complete resistance against target pests. Glyphosate spray assay was carried out for complete removal of weeds. In V1-generation, 70–76% transgenic sugarcane plants were found tolerant against glyphosate spray (3000 mL/ha) under field conditions. While in V2-generation, the replicates of five selected lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were found 100% tolerant against 3000 mL/ha glyphosate spray. It is evident from current study that CEMB-GTGene, CEMB-Cry1Ac and CEMB-Cry2A genes expression in sugarcane variety CPF-246 showed an efficient resistance against cane borers (Chilo infuscatellus) and was also highly tolerant against glyphosate spray. The selected transgenic sugarcane lines showed sustainable resistance against cane borer and glyphosate spray can be further exploited at farmer’s field level after fulfilling the biosafety requirements to boost the sugarcane production in the country.

www.nature.com/scientificreports/ as V 1 generation) were further screened for integration and expression of foreign genes by gene specific PCR, dipstick assay, southern blotting and enzyme linked immunosorbent assay (ELISA). After molecular characterization, qualitative assessment of transgenic plants of V 0 , V 1 and V 2 generation was performed via biotoxicity assay and Glyphosate spray assay.
Screening through polymerase chain reaction (PCR). Genomic DNA was extracted from fresh leaves of transgenic lines using the method described by Edward et al. 38 . After qualitative as well as quantitative assessment of DNA, PCR reactions were set up using CEMB-CrylAc, CEMB-Cry2A and CEMB-GTGene specific primers. DNA from non-transformed plant was included as a negative control while plant expression constructs were included as positive controls. Each PCR reaction was carried out in a total volume of 20 µL having 100 ng of template DNA, 50 pm of each forward and reverse primers, 200 mM dNTPs and 1-2 units of Taq DNA polymerase. Success and specificity of reaction was checked by running 15 µL of each reaction on 1% agarose gel. Primer sequences are given in Supplementary material (Table 1).
Southern blotting. Southern KpnI and HindIII for CEMB-Cry1Ac and CEMB-Cry2A. After complete digestion, each digestion mixture was resolved on 1% agarose gel at 15 V for 18 h. and transferred onto the nylon membrane (Hi-bond) using standard capillary transfer 39 . DIG labeled probe of each gene was prepared using Decanucleotide Biotin labeling Kit (Cat # Ko561) according to the manufacturer's instructions and then hybridization and detection was done using Cat# K0562.
Dipstick assay. Total protein was extracted from fresh leaves of transgenic plants and then quantified as described by Qamar et al. 35 . The GTGene, Cry1Ac and Cry2A expression was detected with EnviroLogix ® dipstick kits (Cat # AS011LSS; Cat # AS003 # CTLSS and Cat # AS005 LSS) following the manufacturer's instructions. Dipsticks were coated with monoclonal antibodies IgG of purified EPSPS, Cry1Ac and Cry2A respectively. The antibody coated dipsticks were dipped in transgenic plants protein samples and incubated at room temperature for 15 min.

Enzyme linked immunosorbant assay (ELISA). Protein expressions of CEMB-Cry1Ac, CEMB-Cry2A,
and CEMB-GTGene were quantified in sugarcane positive lines through ELISA. Total crude protein from fresh leaves was used in ELISA. Total crude protein was extracted using protein extraction buffer (10% Glycerol, 40 mM EDTA (pH 7.5), 100 mM NaCl, 10 mM Tris, 100 mM NH4Cl, 20 mM DTT, 2 mM PMSF) and then quantified by Bradford assay 40 . ELISA was performed according to the instruction manual of Envirologix Kit (Portland USA; Cat # 051) and its results were quantified using Micro Plate ELISA Reader Model ELx800. Serial dilutions of the calibrator were used to generate standard curve by plotting OD 450 of each dilution on Y-axis and corresponding concentration on X-axis. The concentrations of trans-proteins were determined by finding their OD 450 values on the curve and relating them to the corresponding concentration on X-axis.
Leaf biotoxicity assay. Leaf biotoxicity assay was used to demonstrate toxic effect of Cry1Ac and Cry2A encoded endotoxins on larvae of shoot borer chilo infuscatellus. Leaves from transgenic as well as control plants at the age of 20, 40, 60 and 80 days were used for the biotoxicity assays. Leaves from each plant were used to feed three 2nd instar larvae in a petri dish. Each experiment was performed in triplicates. After 3 days, rate of mortality was measured with reference to control.
Glyphosate was sprayed at the optimized rate of 1200 mL/80 L H 2 O/acre on the sugarcane transgenic plants of V 0 as well as V 1 and V2 generations during field study. Same concentration of spray was also used on non-transgenic plants along with weeds present in the sugarcane field.     Figure 3A,B). After confirmation, maxi-prep of the plasmids was done (Supplementary Figure 3C). Selection of sugarcane variety for transformation and field study. In this study, SPF-213, SPF-234, HSF-240 and CPF-246 sugarcane varieties were selected on the basis of their cane yield (t/ha) and sugar recovery (%) for sugarcane tissue culturing and transformation (Supplementary Figure 5), however, after optimization of glyphosate spray assays the best responded variety was selected for further field studies. Biolistic transformation in sugarcane and selection of transgenic plants. Tungsten particles coated with pCEMB-SC12 and CEMB-GTGene plasmid constructs were used for Biolistic transformation. The CEMB-CrylAc + CEMB-Cry2A and CEMB-GTG genes driven by Maize Ubiquitin-1 promoter with kanamycin as selection marker, were successfully transformed into four sugarcane varieties SPF-213, SPF-234, HSF-240, and CPF-246 by using CEMB homemade gene gun (Fig. 3). A total of 400 calli were used for the transformation process (        Table 6). The proteins from putative transgenic sugarcane plants were coated in the ELISA plates. The detections of the respective transprotein from transformed plants were confirmed after specific antibodies with the appearance of yellow color. Quantification of CEMB-Bt. and CEMB-GTGene proteins of 15 clones was done through ELISA, maximum 0.475 µg/g protein of CEMB-Cry1Ac from fresh leaves protein was acquired, 0.567 µg/g CEMB-Cry2A of fresh leaves protein was obtained, while for CEMB-GTGene maximum 0.486 µg/g of total leaf protein was acquired as represented in (Supplementary Table 7 Table 8).

Leaf bio-toxicity assay of transgenic sugarcane plants in V 1 -generation. For further evaluation,
these sugarcane transgenic lines were subjected to leaf bioassay against cane borer Chilo infuscatellus larvae (Fig. 17). Larvae of three different instars from Chilo infuscatellus were permitted, in three replicates, to feed on non-transgenic and transgenic plant leaves. The time intervals of 20, 40, 60 and 80 days were same as in- www.nature.com/scientificreports/ V 0 -generation after plantation in field soil. After 3 days of leaf bioassay, data was composed by counting the alive and dead insect larvae alongside assessing the leaf damages done by these larvae during this experimental time period (Supplementary material Table 9). Up to 70 to 100 (%) mortality of Chilo infuscatellus larvae was resulted from the expressed toxins of these transgenic sugarcane plants, according to variation in the toxins quantity which were observed from these lines during the course of time. Moreover, in control plates, the non transgenic leaves were totally damaged, eaten up and all the larvae were alived and healthy. Different statistical analyses such as Analysis of Variance (ANOVA), Least Significant Difference test (LSD) and Dunnett's test were applied for toxicity-significance-analysis of transgenic sugarcane lines in comparison with non transgenic plant leaves. ANOVA showed that all the sugarcane transgenic lines significantly differ from the non transgenic plants and Dunnett's (Supplementary material Table 9) results demonstrated that five (05) transgenic lines from V 1 -geneartion of CPF-246 are considerably differ from non-transgenic plants, as well as from other transgenic sugarcane lines (within the group) i.e. 4L/2, 5L/5,6L/5, L8/4 and L9/6 lines with respect to their toxicity expression against Chilo Infuscatellus insects.

Discussion
High yielding is the ultimate objective of the crop production including sugarcane 1,35,42,43 . The major constraints to sugarcane are cane borer insects, weeds, drought stress and different viruses 2,5,44 . The present study was aimed to control the cane borers and weeds through the genetic modification of sugarcane with codon optimized insect resistant (CEMB-Cry1Ac + CEMB-Cry2A) and glyphosate tolerant genes (CEMB-GTGene). www.nature.com/scientificreports/ The agro-climatic environment of the Pakistan is relatively encouraging for the sugarcane cultivation. In spite of worldwide area wise fifth position, Pakistan stands at fifteenth position in sugar production which is lowest yield per unit area. In monocots, a stable reproducible transformation method has been very important 45,46 and in sugarcane 44,47 . In the current study, an effort was made for the development of an efficient procedure for its regeneration from calli and inbuilt resistance/tolerance in sugarcane transgenic plants against cane borers and glyphosate herbicide. For this purpose, four local elite varieties CPF-213, CPF-234, HSF-240, and CPF-246 were selected for establishing tissue culture experiments. Young immature leaves were used as explants for calli induction. Immature leaves were found excellent explants for embryogenic callus formation 48 , which is compulsory for genetic modification of sugarcane and also strengthen the genetic transformation procedure in the sugarcane 49 . The embryogenic calli of all the four varieties were obtained on a callus formation media containing 2, 4-D 50,51 . This media was supplemented with casein for the enhancement of embryogenic potential of sugarcane calli 52,53 .    www.nature.com/scientificreports/ The calli were regenerated into micro-shoots, and after the multiplication of these micro-shoots, root intiation was started. These four varieties gave different responses for tissue culturing with reference to callogenesis and regeneration due to genotypic variability among cultivars 37,54 . It was also observed that increased concentration of 2,4-D, more than 4 mg/L, in callus induction media significantly adversely affected the regeneration efficiency of the calli. On increased concentrations, complete loss of the regeneration potential of these calli was noted. These results were consistent with the study by Chengalrayan et al. 55 , who described that fresh biomass of the calli reduced on adding upto 6 mg/L of 2,4-D in the media. All the four varieties were critically judged/screened through tissue culture response 2,50 , and through transformation efficiency on the basis of calli survived (%) and calli regenerated (%) on selection media and finally through glyphosate field spray assays. After through screening, CPF-246 variety was selected for the next generation field studies. The results of tissue culture studies proved CPF-246 (callus induction 90%, regeneration 99%    www.nature.com/scientificreports/ and multiplication 100%) as an excellent variety. Bakhsh et al. 56 also selected varieties for genetic modification by means of regeneration response. Crop genetic modification through the introduction of insecticidal (Cry1Ac, Cry2A) and glyphosate tolerant (GTG) genes is more advantageous over conventional breeding techniques 57 .

CEMB-GTGene
The transgenic sugarcane provides resistance against the cane borer pests with Bt. genes 5,58 . More than 100 Bt. genes have been sequenced which were highly dissimilar in amino-acid sequence and in their few biochemical properties as well. Studies proved that in these toxins' combination, Cry1Ac with Cry2A is better against borer insects or lepidopteran insects 59,60 . The introduction of double Bt. genes Cry1Ac and Cry2A also represents the gene pyramiding which helps in creating multiple traits in one variety 61 . Presently, a broad spectrum herbicide, glyphosate is the most commonly used herbicide. Glyphosate is a non-selective-herbicide that can stop the growth of all plants along with a wide range of herbs and weeds 62 . Glyphosate inhibits the synthesis of 5-enolpyuvyl-3-phosphoshikimate (EPSPS) during the shikimate metabolic pathway and subsequently stops the synthesizing of three very important amino acid tyrosine, tryptophan and Table 9. Molecular analysis of CPF-246 (L9/6-10) transgenic sugarcane plants from V 2 -generation.   Table 10. Level of pest control and dose assessment of CEMB-Cry1Ac, CEMB Cry2A (µg/g) and CEMB-GTGene (µg/g) from five transgenic sugarcane lines in V 2 -generation.  www.nature.com/scientificreports/ phenylalanine 63 . Many crops for herbicide resistance has been developed formerly, including the non-glyphosate herbicide-tolerant transgenics with 2,4-D, Dicamba, HPPD inhibitors and bar transgenes 64,65 . However, all these crops were resistant against specific kind of herbicide, while transgenics with cp4EPSPS are not confined with single kind of herbicid 27,66 . Up till now, nine glyphosate-resistant crops have been introduced, comprising soybean, wheat, canola, polish canola, corn, alfalfa, sugar beet, creeping bent-grass and cotton 17,37 .

CEMB-GTGene
The modified sequences of CEMB for Bt. (CEMB-Cry1Ac + CEMB-Cry2A) and glyphosate tolerant (cp4EP-SPS) genes were used for this transformation study. The nuclear encoded chimeric genes were used in preliminary efforts for the expression of Bt. toxins but the resulted expression level was very low 67 . It was supposed that Bt. genes are AT-rich in comparison with plant genes and therefore led to the consideration that the reasons for low expression can be termination of pre-mature transcription, abnormal mRNA splicing, instable mRNA or incompetent codon usage 68 . Synthetic Bt. toxin genes were created (designed), constructed and cloned to neglect all these undesirable aspects, and the resulted expression of these transgenes in plants was significantly enhanced 69,70 . This jointly with experimenting a variety of promoters and other sequences, led to a considerable development of insect borer resistance and improvement in expressing the toxin levels 0.8% of the overall leaf protein 71 .
In the current study we have constructed and introduced three codon optimized synthetic genes named as CEMB-Cry1Ac + CEMB-Cry2A and CEMB-GTGene in the nuclear genome of sugarcane. The expression of these synthetic genes is under the control of maize Ubiquition-1 promoter 37 . Simple and minimal plasmid cassettes were constructed for maximum protein expression 72,73 . A study was made on comparison between expression of Bt. and GTG genes through two different promoters CAMV35S and Maize Ubiquitin-1 (data not included). The resulted expression under Ubiquitin promoter confirmed higher toxin levels 70 . So maize Ubiquitin promoter was used to enhance the expression of these transgenes and improved increased resistance against cane bores and tolerance for glyphosate herbicide. After transformation a number of transgenic sugarcane plants were regenerated from transformed calli using this plant expression constructs (pCEMB-SC12) for CEMB-Cry1Ac + CEMB-Cry2A and pCEMB-SGTG for glyphosate tolerant gene (CEMB-GTGene).
In this present study the stability and inheritance of three transgenes up to three vegetative generations (V 0 , V 1 , and V 2 ) verified that this technique of transformation can be used efficiently 2 . From 100 transformed calli, 81% was survived on the selection pressure (Kanamycine 50 mg/L) while 48% transformed calli were regenerated on double selection medium (Kanamycine 50 mg/L + glyphosate 50 mM). From 1000 resistant multiplied plants (CPF-246), 15 plants were positive for CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTGene, resulting transformation efficiency 1.5% after optimized glyphosate spray assay (1200 mL/80 L/acre). In monocots, the transformation efficiency was reported from 1 to 5% 30 while co-integration of combine two genes was 85% when a combination of 14 diverse (pUC-based) plasmids was transformed 74   The integration studies of these three transgenes in the genome of the sugarcane were confirmed by Southern DNA hybridization. Both single and double restriction digestion of the plasmids pCEMB-SC12 and pCEMB-SGTG was performed to digest the full cassettes and for having an estimation of the copy numbers. In current study 1-2 copy numbers were found. There are many reports for 1-5 copy numbers of transgenes introduced into crop plants by gene gun method in monocots 76,77 . For V 1 -generation, most of the time there was a co-relation between Cry1Ac and Cry2A banding pattern and intensity of the hybridizing bands, indicative of similar copy numbers of the transgenes. This recommends that the integrated plasmids, in general, integrate as a whole unit 77 . There were no rearrangements detections for these transgenes, Southern DNA hybridization confirming the integration of intact transgenes copies. Komari 78 reported 1-6 copies of integrated genes while majority of the transgenics consist of 1-2 copies.
Transprotein expression levels were confirmed by the ELISA and Dipstick assay. The results of Dipstick-ELISA assay or immuno-Strip assay and quantification through DAS-ELISA confirmed that all of the 15 clones (V 0 ) were consisting of CEMB-Bt. and CEMB-GTGene genes, although the expression levels of these transgenes were variable 79 . There might be many reasons for this, such as variability in transgene copy number 80 , transgene insertion locations in the genome 8 or heterozygosity of the sugarcane transgenic lines, internal as well as external environment 81 . Koncz et al. 82 and Zambryski et al. 83 also reported that in transgenic plants, foreign DNA can integrate randomly on the chromosomal sites in the cells of plants through non-homologous recombination. The highest transprotein expression of CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTGene were observed as 0.475 µg/g, 0.567 µg/g, and 0.486 µg/g respectively. Overall ELISA based comparison CPF-246-(5L/5), CPF-246-(6L/5) were highest in their three expressions and CPF-246 (2L/8) and CPF-246 (5L/1) lines were lowest. The clones of V 0 -generation multiplied with 150 replicates (V 1 -generation) in the field showed positive results for PCR amplification through gene specific primers. Fifteen plants which were randomly selected from V1 and V 2 -generation showed stability for the integration and in protein expressions of CEMB-Cry1Ac, CEMB-Cry2Ac and CEMB-GTGene. The highest transprotein expressions of CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTGene were observed as 0.687 µg/g, 0.611 µg/g, and 0.589 µg/g respectively. Overall ELISA based comparison CPF-246-(5L/5), CPF-246-(6L/5) were highest in their three expressions and CPF-246-(2L/8) and CPF-246 (5L/1) lines were lowest. The stable integration of these transgenes was confirmed through Southern Blot analysis. These results showed similarity to those achieved by Bashir et al. 84 , for transgenic Basmati rice. These results were similar to those obtained by Kiani et al. 85 while studying the expression of Cry1Ac protein through ELISA in transgenic cotton plants. Deng et al. 86 observed similar quantified values in the transgenic lines of rice. In V 2 -generation, screened five transgenic sugarcane lines as 50 clones were multiplied in the field. Molecular analyses of three transgenes confirmed its stable trans-protein expression. The clones of these five lines were positive for all the three transgenes by PCR amplification, Southern Blotting, Dipstick assay and ELISA. All the sugarcane clones in this (3rd) generation (V 2 ) were stable in their expressions 87 . The glyphosate tolerant gene expression was stable with respect to their ELISA protein quantification values and as well as by spray assays. The results showed similarity to those found by Leibbrandt et al. 88 and Manickavasagam et al. 89 . Inheritance of integrated genes was studied stable in these three generations. The transprotein of integrated genes in V 0 , V 1 , and V 2 -generations further verified through insect (Chilo infuscatellus) bioassay.
Bio-toxicity leaf assay. Leaf bio-assay was carried out to check the efficacy of transgenes (CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTGene) proteins against Chilo infuscatellus (2nd instar larvae) shoot borer at four different time intervals. The expressional level of toxins from these genes is significantly imprtant as it should be in such sufficient quantitative level at the infestation time that can protect the crop against attack of shoot borers of Lepidopteran. In V 0 -generation, with the passage of time the toxin levels of CEMB-CrylAc and CEMB-Cry2A were decreased and percentage of cane borer damage increased, as the toxin level dropped from 0.475 to 0.252 µg/g for CEMB-Cry1Ac and 0.467 to 0.278 µg/g for CEMB-Cry2A, but all the transgenic lines showed significant insect mortality (60-100%) in comparison with control plants 90 . In V 1 -generation 15 lines with 150 clones were analyzed for leaf bioassay, each transgenic line was significantly differed from non-transgenic plants and performed better against in leaf bio-toxicity assays. Five lines were stable in their transproteins (CEMB-Cry1Ac and CEMB-Cry2A) expression and were also free of any kind of insect attack in the field where as control plants were totally damaged. In bioassay less damage to transgenic plant and complete consumption of nontransgenic plants verified strong defense of transgenic sugarcane against cane borer (Chilo infuscatellus). Similar results were published against borers 5,8,91 .
Analysis of Variance (ANOVA), the Least Significant Difference Test (LSD) and Dunnett's Test confirmed the significant differences for Chilo infuscatellus mortality (%) between the control and transgenic sugarcane plants. ANOVA and Dunnett's test verified highly significant difference at 5% level of significance for transgenic sugarcane line with respect to Chilo infuscattelus mortality. In V 2 -generation insect mortality was highly stable (90%-100). LSD analysis illustrated that transgenic leaf samples from CPF-246 (6L/5-4) at 20-days produced significantly high Chilo infuscatellus larvae mortality percentage (88%) compared with transgenic line (5L/1-9), which showed lowest mortality percentage value (66%). In general this data revealed the significant leaf damage and insect mortality (%) differences in control and transgenic plants respectively and also supported the conclusions of Riaz et al. 2 ; Weng et al. 8 ; Manikandan 58 .
Success of transformation studies depends upon the integration of the desired genes in the genome of the desired plants in addition to its inheritance to the next generation plants. Inheritance of stably integrated genes was studied up to three vegetative generations. Results acquired from PCR, Southern Blot analysis, Dipstick and ELISA (V 0 -generation) clearly confirmed the amplification and integration of three genes in the genome of www.nature.com/scientificreports/ sugarcane plants. Mainly the Southern Blot analysis of V 1 -generation indicated that two copies of transformed genes were present in the genome of sugarcane plants. ELISA analysis, on the other hand also confirmed the actively integrated genes were efficiently transcribed into protein as toxin. The transprotein of integrated genes in V 0 , V 1 and V 2 generation were verified through insect (Chilo infuscatellus) bioassay.
Herbicide spray assay. Weeds affect by limiting the available nutrients to the major cash crops 92 . When glyphosate (3000 mL/ha) sprayed on the transgenic sugarcane plants, after 15 days 160 out of 300 plants was tolerant to glyphosate stress. The rest of the field plants showed necrotic symptoms, expired tissues and weeds were totally dead after this spray application. In the V 1 -generation, 75% of plants were alived and tolerant for glyphosate (3000 mL/ha) while 25 percent non tolerant plants were observed dead in some lines. The low expression may be due to inner or outer environmental conditions. In the V 2 -generation, ten clone plants from each of five transgenic (CPF-246) lines 4L/2, 5L/5, 6L/5, L8/4, and L9/6 were tested. Such comparable results were formerly reported by Joyce et al. 52 . In V 2 -generation all the lines with their clone replicates were tolerant to glyphosate spray (3000 ml/80 L/hectare). Finally, CPF-246 produced five tolerant lines with CEMB glyphosate tolerant gene. These results are in accordance with the previous studies 37, [93][94][95] . It can be concluded that CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTG genes successfully introduced into the genome of the sugarcane CPF-246. Stable expression and insecticidal activity of transgenic plants was confirmed in V 0 , V 1 and V 2 -generations. Molecular analyses confirmed the expression level of toxin in the transgenic lines of V 0 , V 1 and V 2 generations. Transformation of sugarcane with two Bt. and GTGene will open new directions for the development of a high yielding cane borer resistant and glyphosate tolerant sugarcane. These transgenic lines can be further improved with other high sugar yielding characters. The end product can be a transgenic variety with a number of beneficial traits. There is also an international inclination towards economically safe, ecologically and environmentally friendly methods which can enhance the defense mechanism of the crop against pest pathogens 96,97 . Biological insect control methods by using naturally available bacteria, fungi, and viruses received limited acceptance from the users, as their capability to defend plants has commonly been substandard to results achieved by chemical ways. Insecticides have been relatively successful in increasing crop production by minimize the losses rooted by insects, but the use of potentially dangerous chemical sprays and pesticides is disfavored in several countries and even a few compounds deregistered, secondly chemical sprays are not acceptable in sugarcane. The most effectual way of achieving high crop production is to set up the desired resistance into the crop plant by genetic transformation. Now it's possible to create plants that are resistant to insect borers or having multiple novel disease resistant genes which are modified according to the crop genome and adapted for specific soil or environmental conditions.

Conclusion
The five transgenic sugarcane lines (4L/2, 5L/5, 6L/5, L8/4, L9/6) from CPF-246 variety harboring codon optimized CEMB-Cry1Ac, CEMB-Cry2A and CEMB-GTGene have revealed excellent potential up to 100% mortality of Chilo infuscattellus larvae (Lepidopteran) at the plant age of 80 days along with complete weed removal on 3000 mL/ha glyphosate tolerance level. This study concludes that if approved by the biosafety committee, this transgenic sugarcane can be a good starting material for the farmer's community for the cost effective control of insects and weeds. Further studies are recommended to increase the stable expression of Bt. toxins up to the maturity, which can be achieved by further modification of the gene constructs. It has also been reported that glyphosate crops with tolerance level up to 5000 mL/ha might be more useful to control all types of sugarcane weeds in all the agroclimatic regions of the country. To achieve the above goals new vegetative genes such as Vip3A and Vip3B from Bt. in combination with CEMB-Cry1Ac and CEMB-Cry2A can be more effective, with different enhancers, promoters etc. can be developed for further strengthening/safeguarding this industrial cash crop from the insects and weeds.