Abstract: Agriculture is the backbone of economy of Pakistan
and cotton is the major agricultural export and supreme source of raw
fiber for our textile industry. To combat severe problems of insect
and weed, combination of three genes namely Cry1Ac, Cry2A and
EPSPS genes was transferred in locally cultivated cotton variety
MNH-786 with the use of Agrobacterium mediated genetic
transformation. The present study focused on the molecular screening
of transgenic cotton plants at T3 generation in order to confirm
integration and expression of all three genes (Cry1Ac, Cry2A and
EPSP synthase) into the cotton genome. Initially, glyphosate spray
assay was used for screening of transgenic cotton plants containing
EPSP synthase gene at T3 generation. Transgenic cotton plants which
were healthy and showed no damage on leaves were selected after 07
days of spray. For molecular analysis of transgenic cotton plants in
the laboratory, the genomic DNA of these transgenic cotton plants
were isolated and subjected to amplification of the three genes. Thus,
seventeen out of twenty (Cry1Ac gene), ten out of twenty (Cry2A
gene) and all twenty (EPSP synthase gene) were produced positive
amplification. On the base of PCR amplification, ten transgenic plant
samples were subjected to protein expression analysis through
ELISA. The results showed that eight out of ten plants were actively
expressing the three transgenes. Real-time PCR was also done to
quantify the mRNA expression levels of Cry1Ac and EPSP synthase
gene. Finally, eight plants were confirmed for the presence and active
expression of all three genes at T3 generation.
Abstract: Environmental and toxicological characteristics of formulated pesticides may substantially differ from those of their active ingredients or other components alone. This phenomenon is demonstrated in the case of the herbicide active ingredient glyphosate. Due to its extensive application, this active ingredient was found in surface and ground water samples collected in Békés County, Hungary, in the concentration range of 0.54–0.98 ng/ml. The occurrence of glyphosate appeared to be somewhat higher at areas under intensive agriculture, industrial activities and public road services, but the compound was detected at areas under organic (ecological) farming or natural grasslands, indicating environmental mobility. Increased toxicity of the formulated herbicide product Roundup compared to that of glyphosate was observed on the indicator aquatic organism Daphnia magna Straus. Acute LC50 values of Roundup and its formulating adjuvant polyethoxylated tallowamine (POEA) exceeded 20 and 3.1 mg/ml, respectively, while that of glyphosate (as isopropyl salt) was found to be substantially lower (690-900 mg/ml) showing good agreement with literature data. Cytotoxicity of Roundup, POEA and glyphosate has been determined on the neuroectodermal cell line, NE-4C measured both by cell viability test and holographic microscopy. Acute toxicity (LC50) of Roundup, POEA and glyphosate on NE-4C cells was found to be 0.013±0.002%, 0.017±0.009% and 6.46±2.25%, respectively (in equivalents of diluted Roundup solution), corresponding to 0.022±0.003 and 53.1±18.5 mg/ml for POEA and glyphosate, respectively, indicating no statistical difference between Roundup and POEA and 2.5 orders of magnitude difference between these and glyphosate. The same order of cellular toxicity seen in average cell area has been indicated under quantitative cell visualization. The results indicate that toxicity of the formulated herbicide is caused by the formulating agent, but in some parameters toxicological synergy occurs between POEA and glyphosate.