Tuberculosis (TB) is a life threatening infectious disease which is prevalent throughout the worl... more Tuberculosis (TB) is a life threatening infectious disease which is prevalent throughout the world. Mycobacterium bovis based Bacille Calmette-Gue´rin (BCG) is the only vaccine available against TB however, this (single) vaccine is not enough to eradicate it. Furthermore, numbers of researches from different parts of the World have shown its efficacy as variable. Hence other (better) vaccines like DNA vaccines are needed in addition to BCG in order to achieve desired goal of TB eradication. The current study was aimed to develop subunit based DNA vaccines against TB and to check their efficacy. Two constructs Bfrb-pND14 and Mpt32-pND14 were made and used as DNA vaccines. Endotoxin free DNA preparations were made and used in immunization studies. Twenty Balb/c female mice of age eight weeks were used in trial. Two experimental groups each comprising eight animals were used to inoculate Mpt32-pND14 and Bfrb-pND14 vaccines respectively. A group of four animals was used as negative cont...
This study was conducted to investigate the incidence of malarial infections in human population ... more This study was conducted to investigate the incidence of malarial infections in human population in Tehsil (Yazman) of district Bahawalpur, Punjab, Pakistan. Malarial parasites were identified in the blood slides of suspected patients of the disease from July, 2012 to June, 2013. Out of 974 suspected cases of malaria, 33 (3.38%) were found to be positive in blood smear slides. Out of positive cases, 20 (60.60%) were identified as Plasmodiumvivax, 13 (39.40%) cases with Plasmodium falciparum. Statistically there found no significant difference (P>.05) month wise and gender wise i.e. male and female, while the difference among age group was highly significant (P<.001). It can be concluded that malaria still exist in different areas of Pakistan; can turn to epidemic whenever favorable situations it will have. [SYLWAN., 159(3)]. ISI Indexed 470
The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in th... more The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.
The efficacy of a plant-based vaccine produced in maize by expressing F and HN immunogenic protei... more The efficacy of a plant-based vaccine produced in maize by expressing F and HN immunogenic proteins under a seed-specific promoter against NDV genotype VII was assessed using cell-mediated responses in chickens. Using real-time PCR, the mRNA expression of F and HN PCR confirmed maize plants was compared to non-transgenic control plants on a relative basis. F and HN genes mRNA expression was found to be 20 and 60 times higher in corn seeds than in leaves, respectively. In maize seeds, 35 ug/ml of F protein, or 0.6 percent of total protein, and 41 ug/ml of HN protein, or 0.85 percent of total protein, were found using ELISA. The boost in cell-mediated immune response was evaluated in chickens fed with transgenic corn seeds in comparison with control groups. The qPCR analysis showed an increase in the expression of Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), interleukin-15 (IL-15), Interferon-α, Interferon-β, Interfero...
Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and w... more Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to...
Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmi... more Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmitted by whitefly in persistent circulative manner. In Pakistan, CLCuV causes severe damage to Gossypium hirsutum whereas G. arboreum is resistant to this virus. A total of 86 articles were included in this study that were searched through web of knowledge, web of science and google scholar by using the keywords of cotton, CLCuV, waxes, Begomoviruses and transgenic techniques used against Begomoviruses in plants. Various transgenic strategies i.e., pathogen derived resistance (with and without protein expression) and non - pathogen derived resistance have been adopted to control this virus or its vector. Beside these, some natural defense mechanisms of plant also protect it against the vector. The cuticular waxes make the insects’ attachment difficult to plant surfaces and act as a physical barrier. The cuticular waxes in G. arboreum act as first line of defense against whiteflies and thu...
ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its... more ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g−1, 2.534 µg g−1 and 3.58 µg-g−1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare−1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.
Background: Transgenic technology reflects the incorporation of novel useful traits in crop plan... more Background: Transgenic technology reflects the incorporation of novel useful traits in crop plants like cotton for economic benefits by overcoming the problems including insects’ pests and weeds in special. The present study is the success story of the continuous effort of CEMB team started back in the 1990s. Methods: This study includes characterization of a large number of Bacillus thuringiensis (Bt) strains taken from local soil and subjected to direct transformation of isolated BT genes into local cotton cultivars. Protocols for transformation into cotton plants were optimized and validated by the development of double gene codon optimized (Cry1Ac and Cry2A) transgenic cotton varieties. Results: The resulting GMOs in the form of CEMB-33, CA-12, CEMB-66 have been approved by Punjab Seed Council in 2013 and 2016 respectively. Double Bt and weedicide resistant cotton harboring CEMB-Modified and codon optimized cp4EPSPS (GTGene). These varieties can tolerate glyphosate spray ...
Background: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that a... more Background: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to Cotton Leaf Curl Virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading Gossypium hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length. Methods and Results: To test this hypothesis, G. arboretum was transformed with a PIP2 gene construct (35SCpPIP2)using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple l...
Every year the growth of the poultry industry is severely threatened by a number of infectious vi... more Every year the growth of the poultry industry is severely threatened by a number of infectious viral, bacterial and parasitic diseases. There are a number of vaccines to control these diseases including inactivated virus vaccines, attenuated virus vaccines, live virus vaccines, and subunit vaccines, but they are often relatively expensive and require cold storage and trained people to administer them, especially in developing countries. Plant-based vaccines provide a better option to control these diseases in low profit margin poultry industry. Still there are some challenges in the field of plant-based, so called ‘green’ vaccines. Injection-based oral priming is a big challenge for commercialisation of green vaccines so, new techniques are needed in the field of plant-based vaccine to pass these barriers for commercialisation. This discusses the potential for plant-based vaccines and whether they are good option to control poultry diseases.
Tuberculosis (TB) is a life threatening infectious disease which is prevalent throughout the worl... more Tuberculosis (TB) is a life threatening infectious disease which is prevalent throughout the world. Mycobacterium bovis based Bacille Calmette-Gue´rin (BCG) is the only vaccine available against TB however, this (single) vaccine is not enough to eradicate it. Furthermore, numbers of researches from different parts of the World have shown its efficacy as variable. Hence other (better) vaccines like DNA vaccines are needed in addition to BCG in order to achieve desired goal of TB eradication. The current study was aimed to develop subunit based DNA vaccines against TB and to check their efficacy. Two constructs Bfrb-pND14 and Mpt32-pND14 were made and used as DNA vaccines. Endotoxin free DNA preparations were made and used in immunization studies. Twenty Balb/c female mice of age eight weeks were used in trial. Two experimental groups each comprising eight animals were used to inoculate Mpt32-pND14 and Bfrb-pND14 vaccines respectively. A group of four animals was used as negative cont...
This study was conducted to investigate the incidence of malarial infections in human population ... more This study was conducted to investigate the incidence of malarial infections in human population in Tehsil (Yazman) of district Bahawalpur, Punjab, Pakistan. Malarial parasites were identified in the blood slides of suspected patients of the disease from July, 2012 to June, 2013. Out of 974 suspected cases of malaria, 33 (3.38%) were found to be positive in blood smear slides. Out of positive cases, 20 (60.60%) were identified as Plasmodiumvivax, 13 (39.40%) cases with Plasmodium falciparum. Statistically there found no significant difference (P>.05) month wise and gender wise i.e. male and female, while the difference among age group was highly significant (P<.001). It can be concluded that malaria still exist in different areas of Pakistan; can turn to epidemic whenever favorable situations it will have. [SYLWAN., 159(3)]. ISI Indexed 470
The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in th... more The efficacy of Bt crystal proteins has been compromised due to their extensive utilization in the field. The second-generation Bt vegetative insecticidal proteins could be the best-suited alternative to combat resistance build-up due to their broad range affinity with midgut receptors of insects. The codon-optimized synthetic vegetative insecticidal proteins (Vip3Aa) gene under the control of CaMV35S promoter was transformed into a locally developed transgenic cotton variety (CKC-01) expressing cry1Ac and cry2A genes. Transformation efficiency of 1.63% was recorded. The highest Vip3Aa expression (51.98-fold) was found in MS3 transgenic cotton plant. Maximum Vip3Aa protein concentration (4.23 µg/mL) was calculated in transgenic cotton plant MS3 through ELISA. The transgenic cotton plant (MS3) showed one copy number on both chromatids in the homozygous form at chromosome 8 at the telophase stage. Almost 99% mortality of H. armigera was recorded in transgenic cotton plants expressing double crystal proteins pyramided with Vip3Aa gene as contrasted to transgenic cotton plant expressing only double crystal protein with 70% mortality. The results obtained during this study suggest that the combination of Bt cry1Ac, cry2A, and Vip3Aa toxins is the best possible alternative approach to combat chewing insects.
The efficacy of a plant-based vaccine produced in maize by expressing F and HN immunogenic protei... more The efficacy of a plant-based vaccine produced in maize by expressing F and HN immunogenic proteins under a seed-specific promoter against NDV genotype VII was assessed using cell-mediated responses in chickens. Using real-time PCR, the mRNA expression of F and HN PCR confirmed maize plants was compared to non-transgenic control plants on a relative basis. F and HN genes mRNA expression was found to be 20 and 60 times higher in corn seeds than in leaves, respectively. In maize seeds, 35 ug/ml of F protein, or 0.6 percent of total protein, and 41 ug/ml of HN protein, or 0.85 percent of total protein, were found using ELISA. The boost in cell-mediated immune response was evaluated in chickens fed with transgenic corn seeds in comparison with control groups. The qPCR analysis showed an increase in the expression of Interleukin-1 (IL-1), Interleukin-2 (IL-2), Interleukin-6 (IL-6), Interleukin-8 (IL-8), Interleukin-10 (IL-10), interleukin-15 (IL-15), Interferon-α, Interferon-β, Interfero...
Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and w... more Stacking multiple genes into cotton crop to cop up multiple biotic stresses such as insects and weeds is a promising tool to save crop from losses. Transgenic cotton variety, VH-289, with double Bt and cp4EPSPS genes under the control of 35S promoter was used for the expression analyses and biosafety studies. The transgenic cotton plants were screened through PCR amplification of fragments, 1.7 kb for Cry1Ac, 582 bp for Cry2A and 250 bp for cp4EPSPS; which confirmed the presence of all genes transformed in transgenic cotton. The Cry1Ac + Cry2A and cp4EPSPS proteins were quantified through ELISA in transgenic cotton plants. The Glyphosate assay performed by spraying 1900 mL per acre of glyphosate Roundup further confirmed complete survival of transgenic cotton plants as compared to the non-transgenic cotton plants and all weeds. Similarly, insect infestation data determined that almost 99% insect mortality was observed in controlled field grown transgenic cotton plants as compared to...
Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmi... more Cotton leaf curl virus (CLCuV) is economically important monopartite Geminivirus which is transmitted by whitefly in persistent circulative manner. In Pakistan, CLCuV causes severe damage to Gossypium hirsutum whereas G. arboreum is resistant to this virus. A total of 86 articles were included in this study that were searched through web of knowledge, web of science and google scholar by using the keywords of cotton, CLCuV, waxes, Begomoviruses and transgenic techniques used against Begomoviruses in plants. Various transgenic strategies i.e., pathogen derived resistance (with and without protein expression) and non - pathogen derived resistance have been adopted to control this virus or its vector. Beside these, some natural defense mechanisms of plant also protect it against the vector. The cuticular waxes make the insects’ attachment difficult to plant surfaces and act as a physical barrier. The cuticular waxes in G. arboreum act as first line of defense against whiteflies and thu...
ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its... more ABSTRACT Gossypium arboreum (Desi Cotton) holds a special place in cotton industry because of its inherent ability to withstand drought, salinity, and remarkable resistance to sucking pests and cotton leaf curl virus. However, it suffers yield losses due to weeds and bollworm infestation. Genetic modification of G. arboreum variety FBD-1 was attempted in the current study to combat insect and weedicide resistance by incorporating cry1Ac, cry2A and cp4-EPSPS genes under control of 35S promoter in two different cassettes using kanamycin and GUS as markers through Agrobacterium-mediated shoot apex cut method of cotton transformation. The efficiency of transformation was found to be 1.57%. Amplification of 1700 bp for cry1Ac, 167 bp for cry2A and 111 bp for cp4-EPSPS confirmed the presence of transgenes in cotton plants. The maximum mRNA expression of cry1Ac and cp4-EPSPS was observed in transgenic cotton line L3 while minimum in transgenic cotton line L1. The maximum protein concentrations of Cry1Ac, Cry2A and Cp4-EPSPS of 3.534 µg g−1, 2.534 µg g−1 and 3.58 µg-g−1 respectively were observed for transgenic cotton line L3 as compared to control cotton line. On leaf-feed-based insect bioassay, almost 99% mortality was observed for Helicoverpa armigera on the transgenic cotton plant (L3). It completely survived the 1900 ml hectare−1 glyphosate spray assay as compared to non-transgenic cotton plants. The necrotic spots appeared on the third day, leading to the complete death of control plants on the fifth day of assay. The successful multiple gene-stacking in G. arboreum FBD-1 variety could be further used for qualitative improvement of cotton fiber through plant breeding techniques.
Background: Transgenic technology reflects the incorporation of novel useful traits in crop plan... more Background: Transgenic technology reflects the incorporation of novel useful traits in crop plants like cotton for economic benefits by overcoming the problems including insects’ pests and weeds in special. The present study is the success story of the continuous effort of CEMB team started back in the 1990s. Methods: This study includes characterization of a large number of Bacillus thuringiensis (Bt) strains taken from local soil and subjected to direct transformation of isolated BT genes into local cotton cultivars. Protocols for transformation into cotton plants were optimized and validated by the development of double gene codon optimized (Cry1Ac and Cry2A) transgenic cotton varieties. Results: The resulting GMOs in the form of CEMB-33, CA-12, CEMB-66 have been approved by Punjab Seed Council in 2013 and 2016 respectively. Double Bt and weedicide resistant cotton harboring CEMB-Modified and codon optimized cp4EPSPS (GTGene). These varieties can tolerate glyphosate spray ...
Background: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that a... more Background: Gossypium arboreum is a cotton crop native to tropical and subtropical regions that are naturally resistant to Cotton Leaf Curl Virus (CLCuV). However, its cultivation is unfavorable due to the lower quality and shorter fiber length of cotton when compared to the market leading Gossypium hirsutum. Plasma membrane intrinsic protein 2 (PIP2) is an aquaporin responsible for the transport of water and small molecules across cellular membranes. This fluid transport influences cell elongation and cotton fibre development. Hence, increased PIP2 expression may yield plants with enhanced fiber qualities including length. Methods and Results: To test this hypothesis, G. arboretum was transformed with a PIP2 gene construct (35SCpPIP2)using the Agrobacterium-mediated shoot apex cutting method. Relative expression of the CpPIP2 gene in transgenic plants increased up to 35-fold when compared with non-transgenic controls. Transgenic plants displayed a corresponding increase of staple l...
Every year the growth of the poultry industry is severely threatened by a number of infectious vi... more Every year the growth of the poultry industry is severely threatened by a number of infectious viral, bacterial and parasitic diseases. There are a number of vaccines to control these diseases including inactivated virus vaccines, attenuated virus vaccines, live virus vaccines, and subunit vaccines, but they are often relatively expensive and require cold storage and trained people to administer them, especially in developing countries. Plant-based vaccines provide a better option to control these diseases in low profit margin poultry industry. Still there are some challenges in the field of plant-based, so called ‘green’ vaccines. Injection-based oral priming is a big challenge for commercialisation of green vaccines so, new techniques are needed in the field of plant-based vaccine to pass these barriers for commercialisation. This discusses the potential for plant-based vaccines and whether they are good option to control poultry diseases.
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