Cotton is the most important and in-dispensable part of human life and is the backbone of the economy and employment in the world including Pakistan (Muzaffar et al., 2015). The insect problem is a serious threat for cotton causing an estimated $ 645 million a year and yield losses (Awan et al., 2015). Lepidopteron insects are the major problem causing heavy losses with Heliothus species responsible for an estimated $216 million loss (Gutierrez et al., 2013). Pectinophora gossypiella, which is the pink bollworm, is also a serious problem on a smaller scale of the cotton acreage planted (USA) and accounts for an estimated $71 million in direct damage (Kumar and Kiran, 2011). Control of these insect pests is a major expense and therefore a problem for today’s farmers (Pimentel et al., 2014). On an average 6-7 insect treatments per season are applied against these insects (Pedigo et al., 2014). Chemical insecticide use is however limited due to their expense, their persistence in the environment and their escalating rate of application with decreasing effectiveness (Popp et al., 2013).

 

Manual hoeing is a reliable method for removing weeds but this technique is slow, costly and a time-taking process (Pérez-Ruíz et al., 2014). Total weed seed numbers in the soil was also found to rise significantly after shifting from conventional chemical weed control to non-chemical control (Bond and Grundy, 2001). Hence, complete non-chemical methods are not viable or economically sustainable further, glyphosate (N-phosphonomethyl glycine), a herbicide widely used as a control for weeds belongs to herbicide group ‘glycines’. Glyphosate is a broad-spectrum universal herbicide used to kill annual broadleaf weeds and grasses. Glyphosate affects the shikimate metabolic pathway by preventing the synthesis of 5-enolpyrovyle 3-phosphoshikimate (EPSPS). It inhibits the synthesis of three aromatic amino acids including tryptophan, phenylalanine and trypsin (Yamada et al., 2009).

 

However biotechnology provides a tool to control insects and weeds. In particular genetic engineering of plants utilizing plant genes conferring disease and weed resistance offers an alternative to conventional breeding methods for the improved resistance against insects or weeds (Li et al., 2012). Genes encoding antifungal proteins, such as endochitinase, β-1, 3-glucanases and glucose oxidase, or components of signaling pathways involved in the defense response, have for example been used to generate transgenic plants resistant to various plant pathogens (Ijaz and Khan, 2012). Indeed genetic engineering is the major tool for improving crop yield and reducing insect pest damage and other crop related problem. Finding best cotton and other crop varieties for genetic transformation is however vital for any success to use transgenic insect or weed-resistant cotton (Furbank et al., 2015). Selection of such best cotton varieties with respect to transformation efficiency, acclimatization and with better expression of the transgenic is therefore required to save time, money and man power (Zhao et al., 2011).

 

In this study we transformed three genes, CEMB (Centre of Excellence in Molecular Biology) double BT (Cry1Ac and Cry2A) and herbicide resistance gene (cp4EPSPS).The main aim of this study was to measure transformation efficiency, acclimatization capacity and expression between two cultivars of cotton and finding the best germplasm for further modification of problematic traits.

 

1 Results

Approximately one thousand embryos of each cultivar were transformed with both constructs (Bt and cp4EPSPS gene) using the Agrobacterium mediated transformation method. The overall regeneration efficiency of FH-114 was 71% and CIM-598 was 62%. After two month of continuous kanamycin selection, efficiency was reduced to 20% for FH-114and 14% for CIM-598.

 

After continuous one month kanamycin selection, putative transgenic cotton plants were grown on selection free medium for further one month. The acclimatization therapy was started initially for 15 min and then continuous increase of fifteen minutes up-to one month. During the first five days, plants of both cultivars were stable with little bit dehydrated conditions. Between 10-20 days, further loss of 4% plants was observed for FH-114 and 12% for CIM-598.The remaining plants were shifted to soil. FH-114 plants were more physically healthy than CIM-598 at the end of acclimatization process.

 

Genomic DNA was isolated from both cultivars and PCR was performed with gene specific primers.  An expected 450 bp product for Cry1Ac and a 500 bp product for Cry2A were obtained in transgenic plants of both cultivars (Figure 1 and 2). The cp4EPSPS gene was also amplified with gene specific primers and a 350 bp amplification product was identified on an agarose gel stained with ethidium bromide (Figure 3).

 

After transformation into _˜one thousand embryos, only 12 were positive for Cry1Ac in FH-114 and seven in CIM-598. FH-114 was positive for nine plants and CIM-598 for six as for as Cry2A was concerned. Fifteen plants of FH-114 were positive for GTG and 8 of CIM-598 (Only best are shown in the Figure 1, 2 and 3). Transformation efficiency of FH-114 for Cry1Ac was 1.2% and 0.7% for CIM-598. For Cry2A, efficiency was 0.9% and 0.6%, respectively. GTG transformation efficiency was 1.5% and 0.8% respectively (Figure 5). Both varieties were transformed under similar conditions and mediums.

The best positive plants for each gene were subjected to total protein isolation. Protein specific antibodies were used for protein expression in an ELISA detection system for protein quantification (Muzaffar et al, 2015). Highest Cry1Ac, Cry2A and GTG amounts measured were 1.2, 1, 1.3 ng/µl respectively for FH-114 and 0.9, 0.5, 0.9 ng/µl respectively for CIM-598 (Figure 4).

Mortality percentage of Heliothus armigera 2^nd instar larvae was variable after 30, 60 and 90 days due to variation in the level of gene expression. At different time points during plant growth, CIM-598 had lower gene expression and was therefore more susceptible to chewing insect. In comparison, FH-114 plants were fully toxic (Figure 6). Transgenic cotton plants expressing the cp4EPSPS gene were planted into field containment and no application of weed removal was applied for 3 months. After three months, when the cotton field contained different kind of weeds, glyphosate was sprayed at a rate of 1900 ml/acre. Necrosis developed first on non-transgenic plants, which were then died, and later weeds also. However, in comparison to FH-114 plants, which survived, CIM-598 plants were little stunted and dull even some died (Figure 5).

There are many factors which hinder or cause reduction in cotton production (Brévault et al., 2013) but one of the very serious factor along with weeds is insect pest attack (Muzaffar et al., 2015). Insects and weeds are responsible for 20% & 25% loss in cotton respectively (Awan et al., 2015). It has been well documented that insect and weeds can be controlled by gene over expression (Furlong et al., 2013). In this study an effort was made to transfer two BT (Cry1Ac, Cry2A) and one herbicide resistant gene (cp4EPSPS) into two local cotton varieties named FH-114 and CIM-598 through Agrobacterium-mediated transforation. The purpose was not only to transform the genes but mainly to compare these cultivars with respect to their best gene expression for insect and weed control.

 

Transformation of Bt and the cp4EPSPS gene was done by using the shoot apex method as done by Latif (Latif et al, 2015). Transformation efficiency of both cultivars was different under similar condition. FH-114 transformation efficiency was 1.2% and 0.7% and CIM-598 for Cry1Ac and Cry2A was 0.9% and 0.6% respectively. For GTG, it was 1.5% and 0.8%, respectively for each cultivar. Acclimatization capacity of FH-114 was better than for CIM-598. This may be due to the potential of the introduced genes helping to withstand against worse climate conditions (Lawlor and Tezara, 2009). These differences in expression may due to T-DNA transfer rate, insertion points of transferred genes, genetics and vir genes activity during transformation (Rao et al., 2009). Amplification of a 450 bp product for Cry1Ac, 500 bp for Cry2A and 350 bp for GTG confirmed the successful transformation into FH-114 and CIM-598 cotton plants (Kiani et al., 2013). The ELISA results of Bt and GTG also confirmed their expression in cotton plants. Quantification of Cry1Ac, Cry2A and GTG was the maximum for FH-114 which was 1.2, 1, 1.3 ng/µl, respectively. CIM-598 was lower in expression than FH-114 for all three genes. This may be due to the reason of different germplasm may have different expression capacity of foreign genes (Pérez-Ruíz et al., 2014). The transgenic of both cultivars were further subjected to insect bioassay and glyphosate spraying. FH-114 plants showed more toxic effect to insects compared to CIM-598 plants and the negative control. CIM-598 was little bit susceptible to insect attack and stunted growth was observed after 1900 ml/acre spray of Glyphosate (Roundup™). So 100% mortality of insect and dying of weeds confirmed the action of BT and GTG in cotton plants especially for FH-114. CIM-598 was wavered in action despite harboring all three genes with lower expression. The difference in expression capacities of both cultivars may be the ancestors genetics of both cultivars for insect and weeds trait and FH-114 may be descendent from germplasm which is more dominant and have the resistance capacity to insects and weeds.

 

FH-114 harboring double BT and GTG gene holds good potential to combat with serious problems of insects and weeds and may be good assets for local farmers and National breeders to develop further good varieties using this material against insects and weeds which can pave their role in boosting up economy of Pakistan as compared with CIM-598.

 

Two cotton varieties FH-114 and CIM-598 were transformed with Cry1Ac+Cry2A along with cp4EPSPS gene. The seeds of cotton varieties were received from Cotton Research Station Multan, Pakistan. Concentrated H₂SO₄ was used for delinting while sterilization of seeds was done with 5% HgCl₂ and 10% SDS. Seeds were then allowed to germinate at 30^oC in incubator.

 

Cry1Ac+Cry2A and cp4EPSPS genes were transformed in FH-114 and CIM-598 according to Rao (Rao et al., 2011). Two constructs one with the double Bt and other for the cp4EPSP gene were constructed for cotton transformation under the control of the CaMV35S promoter and NOS terminator sequence. Transgenic plants were screened on the antibiotic kanamycin (50 mg/ml) added to the medium and cultivated in the presence of the antibiotic for selection. Putative transgenic plants were the placed in drug free medium for shoot and root formation as reported by Muzaffar (Muzaffar et al, 2015).

 

Two month old putative transgenic plants were shifted to pots from tubes and were exposed to light for 15 min on the first day and then with 15 min increases to light exposure/day over one month. During acclimatization plants were exposed to light from 10 am in the morning and were watered daily.

 

Genomic DNA from putative transgenic plants was isolated according to Lenin (2001). Reaction mixture for PCR contained 100 ng DNA (2 µl), 10×PCR buffer (2 µl), 2.5 mM MgCl₂ (2 µl), 1 mM dNTPs (2 µL),1 pico moles of each primer (2 µl) and 2.5 U Taq DNA polymerase in a total volume of 20 µl. Gene specific primers are shown in table 1. Reactions were carried out in an ABI 9700 thermocycler under the following conditions, initial DNA denaturation at 94^oC for 5 min then 35 cycles of denaturation at 94^oC for 1 min, primer annealing at 51^oC for Cry2A and GTG, while 50^oC for Cry1Ac for 1 min followed by DNA extension at 72^oC for 3 min. After amplification, products were resolved on a 2% agarose gel and visualized under ethidium bromide staining.

An Envirologix Kit (Cat # 051) was used for the Enzyme Linked Immune Sorbent Assay for Cry1Ac,

Cry2A and cp4 EPSPS expression. Leaf samples (1 g) for transgenic cotton plants were subjected to grinding and total crude protein was isolated by using protein isolation buffer (0.5 M EDTA, Glycerol, 5 M NaCl, 2 M Tris-Cl, NH₄Cl, PMSF, DTT).

 

Transgenic plants were subjected to 2^nd instar larva of Helicoverpa armigera to check their toxic level. A total of three leaves from upper, middle and lower part of transgenic cotton plants of 25, 55 and 85 day old were exposed to Helicoverpa armigera larvae after 2-3 days exposure, insect mortality was determined due to feeding larvae on transgenic and non-transgenic cotton plant material.

 

A glyphosate spray assay was applied where a total of 1900 ml/acre glyphosate (Roundup™) was sprayed on both transgenic and non-transgenic cotton plants. Glyphosate was prepared to its final concentration by dissolving the herbicide in water.

 

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