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Öğe AN EFFICIENT AND COST EFFECTIVE STERILIZING METHOD WITH LEAST MICROBIAL CONTAMINATION AND MAXIMUM GERMINATION RATIO FOR IN VITRO COTTON (Gossypium hirsutum L.) CULTURE(PAKISTAN AGRICULTURAL SCIENTISTS FORUM, 2016) Bakhsh, A.; Anayol, E.; Sancak, C.; Ozcan, S.Cotton (Gossypium hirsutum L.) is a highly significant crop because of its quality for textile industry of the world. With the advent of genetic engineering, cotton has gone through several genetic improvements that encode tolerance against biotic and abiotic stresses. The efficient seed sterilization method is essential for successful genetic improvement in cotton. The present study was conducted to compare different sterilizing agents employed for in vitro culture of cotton to find out an efficient, reproducible, cost effective and genotype independent in vitro seed sterilization procedure of cotton. The two cultivated cotton cultivars i.e. GSN-12 and Coker-312 were used for these studies that were further transformed with insecticidal genes under different promoters. The different sterilizing agents used (in separate or combination) were ethanol, commercial bleach (NaOCl), mercuric chloride (HgCl2), Sodium Dodecyle (SDS), hydrogen peroxide (H2O2). The surface sterilized seeds of both cultivars were cultured on MS medium and data was recorded for contamination free seeds and germination ratio. The results showed that 5% n-Hexane in combination with 2% of H2O2 are the best sterilizing agents for both cultivars when compared to the efficacy of other chemicals. Seeds treated with n-hexane and hydrogen peroxide had minimum microbial contamination with improved germination rate. The sterilizing method was successfully used for surface sterilization of other cotton cultivars in our project. According to our knowledge, this is the first report of using n-hexane in combination with H2O2 for surface sterilization of cotton seed for in vitro culture studies.Öğe An efficient and cost effective sterilizing method with least microbial contamination and maximum germination ratio for in vitro cotton (gossypium hirsutum L.) culture(Pakistan Agricultural Scientists Forum, 2016) Bakhsh, A.; Anayol, E.; Sancak, C.; Özcan, S.Cotton (Gossypium hirsutum L.) is a highly significant crop because of its quality for textile industry of the world. With the advent of genetic engineering, cotton has gone through several genetic improvements that encode tolerance against biotic and abiotic stresses. The efficient seed sterilization method is essential for successful genetic improvement in cotton. The present study was conducted to compare different sterilizing agents employed for in vitro culture of cotton to find out an efficient, reproducible, cost effective and genotype independent in vitro seed sterilization procedure of cotton. The two cultivated cotton cultivars i.e. GSN-12 and Coker-312 were used for these studies that were further transformed with insecticidal genes under different promoters. The different sterilizing agents used (in separate or combination) were ethanol, commercial bleach (NaOCl), mercuric chloride (HgCl2), Sodium Dodecyle (SDS), hydrogen peroxide (H2O2). The surface sterilized seeds of both cultivars were cultured on MS medium and data was recorded for contamination free seeds and germination ratio. The results showed that 5% n-Hexane in combination with 2% of H2O2 are the best sterilizing agents for both cultivars when compared to the efficacy of other chemicals. Seeds treated with n-hexane and hydrogen peroxide had minimum microbial contamination with improved germination rate. The sterilizing method was successfully used for surface sterilization of other cotton cultivars in our project. According to our knowledge, this is the first report of using n-hexane in combination with H2O2 for surface sterilization of cotton seed for in vitro culture studies. © 2016, Pakistan Agricultural Scientists Forum. All rights reserved.Öğe Ectopic expression of common bean ERF transcription factorPvERF35promotes salt stress tolerance in tobacco(Wiley, 2020) Kavas, M.; Gokdemir, G.; Secgin, Z.; Bakhsh, A.In the present study, a TINY-like AP2/ERF gene,PvERF35i,was amplified from common bean (Phaseolus vulgarisL.), cloned and functionally characterized by overexpressing in tobacco cv. Petite havana. Transgenic plants overexpressingPvERF35were generated usingAgrobacterium-mediated transformation and used to evaluate the possible roles of the transgene under salt stress conditions. Evaluation of transgenics was completed using both molecular and biochemical analysis. PCR, Southern blot and RT-qPCR assays revealed the correct integration and enhanced expression of the transgene. Physiological and biochemical analysis of transgenic plants showed their better performance compared to the wild type in terms of germination and survival rates and root and shoot growth under salt stress treatment (200 mM NaCl). Having a high concentration of proline, APX and POX, thePvERF35overexpressed plants were physiologically and morphologically less affected by salt stress application.In silicopromoter analysis of thePvERF35gene led to identification of importantcis-regulatory elements, MYB, MYC and TGACG-motif, annotated with salt response of plants. The protein-protein interaction network showed that there was a strong association between ABC transporter proteins and PvERF35 protein. Salt stress-related miRNA, miRNA156 and miRNA159, targetingPvERF35were identified usingin silicotarget finding analysis. These findings suggest thatPvERF35functions as a stress-responsive transcription factor in differential modulation of salt stress tolerance and may have applications in the engineering of economically important crops.Öğe EFFECTS OF PRECONDITIONING, PLANT GROWTH REGULATORS AND KCL ON SHOOT REGENERATION OF PEANUT (ARACHIS HYPOGEA)(PAKISTAN AGRICULTURAL SCIENTISTS FORUM, 2016) Day, S.; Aasim, M.; Bakhsh, A.Peanut (Arachis hypogeae) is an important legume used as green manure and cover crop also. In this study, preconditioned plumular apex explants with 20 mg L-1 BAP or unconditioned explants were cultured for 8 weeks on MS medium having 0.25-2.0 mg/l BAP with or without 0.25 mg L-1 NAA. 100 % shoot regeneration was recorded from both conditioned and unconditioned explants. However, conditioning of explants resulted in 1.5-2.0 fold more number of shoots per explants with relatively shorter shoots compared to unconditioned explants. Shoots per explant from unconditioned explants ranged 1.33-3.93 with 1.86-5.03 cm shoot length. Whereas, shoots per explant and shoot length of preconditioned explants ranged 2.73-5.87 and 0.77-1.75 cm respectively. In second experiment, 10, 15 and 20 muS/cm of KCl in combination with BA-NAA concentrations also resulted in 100 % shoot regeneration. Higher concentration of KCl imposed necrosis on the explants and generated shoots with low leaf area. Increased KCl concentration inhibited the shoots per explant but also enhanced the shoot length. In vitro regenerated shoots from both experiments were rooted successfully using IBA and acclimatized in the pots. Results revealed the superiority of preconditioning on shoot regeneration and KCl for shoot elongation.Öğe Effects of preconditioning, plant growth regulators and Kcl on shoot regeneration of peanut (Arachis hypogea)(Pakistan Agricultural Scientists Forum, 2016) Day, S.; Aasim, M.; Bakhsh, A.Peanut (Arachis hypogeae) is an important legume used as green manure and cover crop also. In this study, preconditioned plumular apex explants with 20 mg L-1 BAP or unconditioned explants were cultured for 8 weeks on MS medium having 0.25-2.0 mg/l BAP with or without 0.25 mg L-1 NAA. 100 % shoot regeneration was recorded from both conditioned and unconditioned explants. However, conditioning of explants resulted in 1.5-2.0 fold more number of shoots per explants with relatively shorter shoots compared to unconditioned explants. Shoots per explant from unconditioned explants ranged 1.33-3.93 with 1.86-5.03 cm shoot length. Whereas, shoots per explant and shoot length of preconditioned explants ranged 2.73-5.87 and 0.77-1.75 cm respectively. In second experiment, 10, 15 and 20 ?S/cm of KCl in combination with BA-NAA concentrations also resulted in 100 % shoot regeneration. Higher concentration of KCl imposed necrosis on the explants and generated shoots with low leaf area. Increased KCl concentration inhibited the shoots per explant but also enhanced the shoot length. In vitro regenerated shoots from both experiments were rooted successfully using IBA and acclimatized in the pots. Results revealed the superiority of preconditioning on shoot regeneration and KCl for shoot elongation. © 2016, Pakistan Agricultural Scientists Forum. All rights reserved.Öğe Elimination of Unnecessary Protein Production of Transgenes in Transgenic Plants(SPRINGER, 2014) Ozcan, S.; Anayol, E.; Bakhsh, A.; Ozcan, S.; Aasim, M.; Gurbuz, B.; Unlu, L.[Abstract Not Available]Öğe Exploring the amenability of onions to Agrobacterium mediated transformation(Pakistan Agricultural Scientists Forum, 2019) Amin, K.; Bakhsh, A.; Gökçe, A.F.The onions exhibit recalcitrant nature when subjected to genetic modifications. The study was conducted to optimize various factors affecting Agrobacterium mediated transformation in onion. Factors such as genotype dependency, type of explants and response of growth regulators were investigated. Agrobacterium strain LBA4404 harboring pBIN19 binary vector was used to infect different explants of two cultivars (Kral and Sampiyon). The T-DNA region of vector contained uidA gene (interrupted by an intronic sequence) to screen primary transgenic shoots at earlier stage and nptII gene as plant selectable marker. Results exhibited the genotype and explant dependency towards genetic modification of both cultivars. Based on the rate of callus induction, regeneration response and GUS histochemical assays, cultivar “Kral” was found better compared to “Sampiyon”. The best callus induction was observed on MS medium supplemented with 1:10 ratio of 2,4-D and BAP respectively. Out of 355 primary transformants, 87 primary transformants were recorded as positive when subjected to PCR assays; 51 belonging to Kral showing tendency of genotype to genetic improvement. Results showed that highest number of putative transgenic plants was contributed by mature embryos followed by seeds and basal plates explants. Onion seed as explant has been used successfully for first time as no evidence in earlier literature was found. The optimization of these factors in Agrobacterium tumefaciens-mediated transformation will provide a gateway to introduce any desired trait in onion. © 2019, Pakistan Agricultural Scientists Forum. All rights reserved.Öğe EXPLORING THE AMENABILITY OF ONIONS TO AGROBACTERIUM MEDIATED TRANSFORMATION(Pakistan Agricultural Scientists Forum, 2019) Amin, K.; Bakhsh, A.; Gokce, A. F.The onions exhibit recalcitrant nature when subjected to genetic modifications. The study was conducted to optimize various factors affecting Agrobacterium mediated transformation in onion. Factors such as genotype dependency, type of explants and response of growth regulators were investigated. Agrobacterium strain LBA4404 harboring pBIN19 binary vector was used to infect different explants of two cultivars (Kral and Sampiyon). The T-DNA region of vector contained uidA gene (interrupted by an intronic sequence) to screen primary transgenic shoots at earlier stage and nptII gene as plant selectable marker. Results exhibited the genotype and explant dependency towards genetic modification of both cultivars. Based on the rate of callus induction, regeneration response and GUS histochemical assays, cultivar Kral was found better compared to Sampiyon. The best callus induction was observed on MS medium supplemented with 1: 10 ratio of 2,4-D and BAP respectively. Out of 355 primary transformants, 87 primary transformants were recorded as positive when subjected to PCR assays; 51 belonging to Kral showing tendency of genotype to genetic improvement. Results showed that highest number of putative transgenic plants was contributed by mature embryos followed by seeds and basal plates explants. Onion seed as explant has been used successfully for first time as no evidence in earlier literature was found. The optimization of these factors in Agrobacterium tumefaciens-mediated transformation will provide a gateway to introduce any desired trait in onion.Öğe Genetic approaches for engineering biotic stress resistance in potato (Solanum tuberosum L.)(Pakistan Agricultural Scientists Forum, 2020) Bakhsh, A.; Dangol, S.D.; Naeem, M.; Azimi, M.H.; Yasmeen, A.Potato is one of the most important food crops in terms of annual production and food security worldwide. The crop is affected by several types of biotic stresses, e.g. insects, viruses, fungus, nematodes and weeds, which are the prominent limiting factors for its production. The conventional breeding methods in potato have been associated with limitations; none of the present day commercial cultivar has built-in resistance against biotic stresses. There is strong need for the development of new resistant potato varieties to cope against biotic stresses using non-classical approaches in combination with classical methods. The scientific literature suggests the contribution of modern biotechnological techniques for the development of transgenic potato lines resistant against insects and diseases. The present comprehensive review describes different genetic engineering approaches for the development of transgenic potatoes resistant to insects, weeds, nematodes, fungus and viruses by fellow researchers worldwide. It also gives an insight into modern technologies, e.g. RNAi and CRISPR-Cas9, which have emerged recently and can be implemented in the development of biotic stress resistant potato cultivars. © 2020, Pakistan Agricultural Scientists Forum. All rights reserved.Öğe GENETIC APPROACHES FOR ENGINEERING BIOTIC STRESS RESISTANCE IN POTATO (Solanum tuberosum L.)(Pakistan Agricultural Scientists Forum, 2020) Bakhsh, A.; Dangol, S. D.; Naeem, M.; Azimi, M. H.; Yasmeen, A.Potato is one of the most important food crops in terms of annual production and food security worldwide. The crop is affected by several types of biotic stresses, e.g. insects, viruses, fungus, nematodes and weeds, which are the prominent limiting factors for its production. The conventional breeding methods in potato have been associated with limitations; none of the present day commercial cultivar has built-in resistance against biotic stresses. There is strong need for the development of new resistant potato varieties to cope against biotic stresses using non-classical approaches in combination with classical methods. The scientific literature suggests the contribution of modern biotechnological techniques for the development of transgenic potato lines resistant against insects and diseases. The present comprehensive review describes different genetic engineering approaches for the development of transgenic potatoes resistant to insects, weeds, nematodes, fungus and viruses by fellow researchers worldwide. It also gives an insight into modern technologies, e.g. RNAi and CRISPR-Cas9, which have emerged recently and can be implemented in the development of biotic stress resistant potato cultivars.Öğe Knock-down of Vital Gene(s) of Tuta absoluta (Meyrick) (Lepidoptra: Gelechiidae) Using in Planta RNAi.(Springer, 2022) Hashmi, Rnai. M. H.; Demirel, U.; Bakhsh, A.[Abstract Not Available]Öğe Knockout of Potato Invertase Inhibitor Gene by CRISPR/CAS9 Based Approach.(Springer, 2019) Dangol, S. D.; Barakate, A.; Caliskan, M. E.; Bakhsh, A.[Abstract Not Available]Öğe Lethal Response of Orally Delivered V-ATPase dsRNA in the Colorado Potato Beetle Leptinotarsa decemlineata.(Springer, 2023) Tariq, H.; Gokce, A.; Hashmi, M. H.; Aksoy, E.; Bakhsh, A.[Abstract Not Available]Öğe Members of Alliaceae; better source of plant lectins to combat resistance against sucking pests of crops(INT SOC HORTICULTURAL SCIENCE, 2016) Bakhsh, A.; Zia, M. A. B.; Hussain, T.; Tekeli, F. O.; Gokce, A. F.; Gokce, AFTransgenic Bt crops were planted on an area of approximately 45 million hectares in combination with herbicide resistance trait in recent years. The available scientific literature does not show any concrete evidence of cry gene exhibiting resistance against sucking pest particularly in commercialized crops. Therefore, the common crop protection practice against sucking pests is the application of chemical insecticides both for transgenic and non transgenic crops. The increased pest status and resistance to chemical insecticides provide impetus for the development of alternative management strategies. Lectins are carbohydrate-binding proteins that specifically recognize glycans of glycoproteins, glycolipids or polysaccharides with high affinity and mediate various cell-cell and host-pathogen interactions, besides innate immune responses. The ability of plant lectins to control sap-sucking insects is considered as most significant as these devastating pests are unresponsive to insecticidal Bt genes and are constant threat to crops by damaging them directly or by acting as vector for different pathogens. Different researchers worldwide have established the successful efficacy of plant lectins against sucking insect pest in transgenic rice, tobacco, oilseed rape, potato, wheat, maize, chickpea and cotton. Among various plant lectins introduced in crops, the monocot mannose binding lectins i.e. garlic (Album sativum L. leaf agglutinin, ASAL), onion (Album cepa L. agglutinin, ACA), snowdrop (Galanthus nivalis L. agglutinin, GNA) has exhibited quite promising and significant resistance to jassids (Amrasca devastans), whiteflies (Bernisia tabaci) and aphids (Aphis gossypii). The present study focuses the research endeavours in various transgenic crops expressing plant lectins gene(s) against sucking pests of crop plants.Öğe MUTATION BREEDING IN POTATO; ENDEAVORS AND CHALLENGES(Pakistan Agricultural Scientists Forum, 2018) Zia, M. A. B.; Bakhsh, A.; Caliskan, M. E.Mutation breeding is a resolute application of mutations to develop the desired characteristics in crops to get increased yield. Since the first release of mutant potato cultivar in Belgium in 1968, mutation breeding in potato found a niche in plant breeding because of the increased quality and yield improvements. Six mutated potato cultivars have been registered from all over the world. Many scientists are working on potato mutation breeding and have reported appreciable results especially quality improvement of the crop, decreased amylose contents, salt and heat tolerance, and resistance to late blight. However conventionally induced mutation has some distinct limitations, especially in the applications of crop-breeding, as percentage of desired mutation is very less in the field; by using in vitro techniques with the combination of routine mutagenesis has overawed this obstruction too. Thus this technique can be the part of the other plant breeding techniques to increase the yield and to nourish the rapidly increasing world's population. The present review focuses the achievements, endeavors and future challenges of mutation breeding in potato.
