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Potato virus Y (PVY) is among the most destructive potato viruses and a solemn threat to efficient seed production worldwide. Almost 70% potato yield losses are due to early PVY infection. Coat protein (CP) gene has a significant function in host adaptation in most of the plant viruses. CP has become more frequent and extensively studied in PVY and other potyviruses to generate resistance against the potyviruses in different crops. In this study, we also targeted the CP gene and evaluate the in vitro potential of Iberis gibraltarica total protein extract against the CP gene expression. Total protein of Iberis gibraltarica, was isolated and quantified. The full length CP gene was amplified from PVY infected potato plant leaves and cloned into mammalian expression vector. It was later transfected into mammalian cell line to obtain the transient expression and mRNA expression of the CP gene against the total protein of Iberis gibraltarica was analyzed through the real-time PCR. Results show that total protein of Iberis gibraltarica down regulates the mRNA expression more than 90% in vitro studies.

Soybean Mosaic Disease (SMD) is one of the most serious, devastating, and widespread diseases of Glycine max (L.) Merry. caused by Soybean Mosaic Virus (SMV). It is more prevalent in low-lying and ...

The RNAi-based insecticide for crop protection has witnessed a rapid improvement over the years. However, its potential to efficiently control maize stem borer (Chilo partellus) pest has remained unexplored. In this study, double-stranded C. partellus chitinases (dsCHI ) toxicity was investigated on C. partellus larvae fed on an artificial diet coated with bacterially-expressed dsCHI and purified dsCHI. Furthermore, we developed transgenic Maize lines expressing dsRNA targeted against C. partellus chitinases transcripts and performed a detached leaf insect feeding bioassays. Our results revealed that the chitinase transcripts expression was significantly downregulated by 55% and 51% in the larvae fed on purified dsCHI at 5- and 15-days. Post-exposure to bacterially-expressed dsCHI resulted in 57% and 82% reduction in chitinase mRNA transcripts after 5- and 15-days. The larvae exhibited various phenotypic distortion levels across the developmental stages, and 53% mortality occurred in transgenic fed larvae compared to those fed on non-transgenic leaves. In conclusion, we have identified C. partellus chitinase gene as a potential target for RNAi mediated control and demonstrated that oral delivery via bacteria and plant-mediated delivery are viable means of achieving C. partellus RNAi mediated control.

The growth and morphological development including fins, spine distribution and pigmentation of larval and juvenile of hatchery‐reared yellow puffer, Chonerhinos naritus were described to provide essential information on the early life history of this species. The total length (TL) of newly hatched larvae was 3.42 ± 0.23 (mean ± SD) mm, reaching 5.66 ± 0.38 mm on 5 days after hatched (DAH), 7.80 ± 0.28 mm on 11 DAH, 9.88 ± 0.40 mm on 27 DAH and 10.92 ± 0.58 mm on 30 DAH. The yolk was completely absorbed in preflexion larvae at 4 DAH. The mouth opening started at 3 DAH of yolk sac larvae, while the teeth appeared starting from preflexion larvae at 7 DAH. Overall aggregate fin ray numbers including caudal fin attained full complement in postflexion larvae at 27 DAH. Several melanophores with appearance of small stellate were first appeared dorsally on the head of flexion larvae at 13 DAH, expanded at the dorsal region of the head, above the eye in juveniles at 30 DAH. The spines first appeared in preflexion larvae of C. naritus at 7 DAH, covering the ventral skin region below pectoral fin base and expanded to the ventral part of the body and nearly covered the whole abdomen region before the anus and below the eyes in juveniles. C. naritus remain as larvae for approximately 29 days, during which they metamorphose to the juvenile stage prior to sexual maturation. Observations in larvae development of C. naritus revealed similar characteristics with other Tetraodontidae species.

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.

Potato (Solanum tuberosum L.), an important food crop in the world, is susceptible to many fungal pathogens including Alternaria solani and Fusarium oxysporum causing Fusarium wilt and early blight diseases. Mycoparasitic fungi like Trichoderma encode chitinases, cell wall degrading enzymes, with high antifungal activity against a wide range of phytopathogenic fungi. In this study, a binary vector harboring endochitinase gene of ~1,000 bp was constructed and used to transform potato nodes through Agrobacterium-mediated transformation. Out of several primary transformants, two transgenic potato lines were verified for transgene insertion and integration by Southern blot. In a pot experiment for Fusarium resistance, the transgenic potato lines didn’t show any symptoms of disease, instead they remained healthy post infection. The transgenic potato lines exhibited 1.5 fold higher mRNA expression of endochitinase at 7 days as compared to 0 day post fungus inoculation. It was evident that the mRNA expression decreased over days of inoculation but was still higher than at 0 day and remained stable upto 30 days post inoculation. Similarly, for A. solani infection assay, the mRNA expression of the endochitinase gene was 3 fold higher 7 days post inoculation compared to expression at 0 day. Although the expression decreased by1.2 fold during subsequent days post infection, it remained stable for 30 days, suggesting that protection in transgenic potato plants against fungal pathogens was achieved through an increase in endochitinase transcript.

RNA silencing is an important antiviral mechanism in plants. Small interfering RNAs (siRNA) or short hairpin RNAs (shRNA) are key stimulators for RNA silencing by acting as executors of viral restriction. Here, we have utilized RNAi technology to suppress potato virus X (PVX) in a transgenic potato cultivar. A stable shRNA of 107 bp directed against a conserved region in the coat protein (CP) gene of PVX was designed with stem and loop sequences derived from a microRNA (miR403; an active regulatory miRNA in potato). The shRNA transgene was directionally cloned in a plant binary vector under the influence of the cauliflower mosaic virus 35S (CaMV35S) constitutive promoter. The pre-shRNA construct was introduced into the potato cultivar Sante through Agrobacterium and transgene insertion was confirmed by testing using PCR (polymerase chain reaction). Upon artificial inoculation of transgenic and non-transgenic potato lines with PVX, variable resistance was revealed through qRT-PCR among the transgenic potato lines. Compared to non-transgenic potato plants, the transgenic potato lines—D5, P3, P9, P14, P21, S11 and S21—showed undetectable levels of CP-PVX mRNA. However, the transgenic lines D4 and P16 exhibited 70% and 75%, respectively, reducing mRNA expression of CP-PVX. The transgenic potato lines remained healthy, with no detectable disease symptoms as compared to the control plants which showed chlorosis and mosaic symptoms. Hence, the expression of virus specific shRNAs is a novel, effective and predictable approach to engineer resistance against PVX in transgenic plants.

The prevalence of insect resistance against Bt toxins has led to the idea of enhancing demethylation from cell wall pectin by pectin methylesterase enzyme for overproduction of methanol which is toxic to insects pests. The AtPME and AnPME fragments ligated into pCAMBIA1301 vector were confirmed through restriction digestion with EcoR1 and BamH1. Excision of 3363 bp fragment from 11,850 bp vector confirmed the ligation of both fragments into pCAMBIA1301 vector. Transformation of pectin methylesterase-producing genes, i.e., AtPME and AnPME from Arabidopsis thaliana and Aspergillus niger cloned in plant expression vector pCAMBIA1301 under 35S promoter into cotton variety CEMB-33 harboring two Bt genes Cry1Ac and Cry2A, respectively, was done by using shoot apex-cut Agrobacterium-mediated transformation method. The plantlets were screened on MS medium supplemented with hygromycin on initial basis. Amplification of 412 and 543 bp, respectively, through gene-specific primer has been obtained which confirmed the successful introduction of pCAMBIA AtPME and AnPME genes into cotton variety CEMB 33. Relative expression of AtPME and AnPME genes through real-time PCR determined the expression level of both gene ranges between 3- and 3.5-fold in different transgenic cotton lines along with quantity of methanol ranging from 0.8 to 0.9% of maximum while 0.5% to 0.6% of minimum but no expression was obtained in negative non-transgenic control cotton plant with least quantity of methanol, i.e., 0.1%. Almost 100% mortality was observed in insect bioassay for Helicoverpa armigera on detached leaves bioassay and 63% for Pink Bollworm (Pectinophora gossypiella) on growing transgenic cotton bolls as compared to positive control transgenic cotton with double Bt genes where mortality was found to be 82% for H. armigera and 50% for P. gossypiella while 0% in negative control non-transgenic plants.

Potato (Solanum tuberosum L.) is considered to be frost-susceptible as short spells of frost can reduce the tuber yield and quality. Ice recrystallization inhibition (IRI) protein helps prevent growth of ice crystals in the cell apoplast during frost and help prevent damage associated with freezing stress. In this study, we investigated the in planta potential of Lolium perenne derived IRI3 transgene in improving the tolerance of transgenic potato lines for freezing stress. The codon optimized IRI3 transgene was introduced into potato cultivar Diamant through Agrobacterium mediated transformation. Three transgenic potato lines were successfully generated which were confirmed for transgene insertion and genomic integration by polymerase chain reaction and Southern blot. It was evident that the IRI3 transcript decreased in initial 24 h of cold stress treatment while the IRI3 mRNA expression up regulated in subsequent hours of cold treatment with maximum increase to 20 folds at 96 h post stress. A similar trend was also revealed in ion-leakage assay which showed that during cold stress, the transgenic potato lines depicted reduced ion leakage of 14-22% as compared to non-transgenic control plants. Further, the generated transgenic potato lines were tolerant to the frost spell in quarantine field conditions as compared to the non-transgenic potato lines. Additionally, the transgenic lines exhibited efficient recovery post frost injury in field conditions. The biochemical profiles of chlorophyll, proline and higher levels of antioxidant enzyme (superoxide dismutase, Catalase) activity and malondialdehyde levels showed that despite the phenotypic impact of low temperature, the transgenic potato lines quickly adjusted to maintain their cellular homeostasis post freezing stress by increasing the antioxidant defenses. This study suggests that up regulation of IRI3 transcript and regulatory network of cold stress response in transgenic potato lines improve frost tolerance and help stabilize yield in cultivated potato.

The aim of this study was to ensure the systematic protein expression of two genes (GTG and Cry1Ac) under the influence of two different constitutive promoters i.e. Ubiquitin-1 and CaMV 35S promoters in different sugarcane lines. PCR amplification of GTG and Cry1Ac was achieved from putative transgenic plants through gene specific primers. Qualitative comparisons of GTG and Cry1Ac genes expression under two different promoters were obtained through protein dot blot and dipstick assay. The appearance of comparatively dark color dots in dot blot and dark color bands on dipstick with Ubiquitin as compared to light color bands with CaMV35S promoter, qualitatively confirmed high protein expression of two genes under Ubiquitin promoter. In quantitative gene expression comparisons maximum optical density (OD) at 450 nm of UV-light was obtained for GTG (3.7 OD) and Cry1Ac (3 OD) under Ubiquitin promoter, while for GTG (1.6 OD) and Cry1Ac (2.5 OD) with CaMV 35S promoter. The results indicated higher expression of two genes under Ubiquitin-1 promoter in sugarcane was found as compared to CaMV 35S promoter. This study provides a guide for stable and high expression of transgenes with reference to Ubiquitin-1 promoter which can be utilize in sugarcane as well as in other monocots.

Phytophthora capsici is the most destructive pathogen of vegetables that has blown a serious threat to chili pepper plants. We discussed the control of Phytophthora capsici through manipulation of genetic architecture of chili plant. Various genes encoding transcriptional regulatory and defense related putative proteins such as pathogen-related protein (PR), Anti-Microbial peptides (AMPs), Polygalacturonase-inhibiting proteins (PGIPs), Lipid Transfer Protein (LTP), Pectin Methylesterase (PME), Leucine-rich repeat proteins (LRRs), Osmotin-like and Thaumatin-like protein, have been identified in Capsicum. We also discussed the enhanced resistance to P. capsici infection by treatment with a variety of abiotic and biotic inducers that act on defense signaling pathways involved in disease resistance. We highlighted the vulnerability of chili crop with reference to its genetic resources against Phytophthora blight. Disease control through chemicals is becoming problematic, so we proposed other ways to control the disease severity.

Plant chitinases are strong antifungal enzymes, and their genes can be utilized to engineer crop plants, including potatoes, with tolerance against fungal pathogens. The purified recombinant chitinase protein significantly inhibited the growth of phytopathogenic fungi, including Alternaria solani, in a qualitative invitro antifungal assay. Furthermore, transgenic potatoes (variety Desiree) over-expressing the endo-chitinase gene were successfully developed using the Agrobacterium-mediated transformation method. The transformation efficiency was 21% based on preliminary molecular and biochemical analyses, including histochemical GUS staining, PCR, and Southern blotting. Crude protein extracts from transgenic potato plants inhibited the hyphal growth of A. solani from 39.5 to 60.5% in a quantitative in vitro assay. The in-vitro bioassay showed that transgenic potato plants exhibit strong resistance against A. solani infection as these plants remains green and healthy, while non-transgenic control plants turned yellow and eventually died 3-week post infection. In the detached leaf assay, the leaves of transgenic plants challenged with A. Soltani showed a decrease in susceptibility compared to the control. The number of necrotic spots in the control leaves was significantly higher compared to the number in the transgenic leaves of potato plants infected with A. solani. Furthermore, A. solani infection triggered a gradual increase in mRNA expression of the transgene in transgenic plants compared to non-transgenic plants as revealed in the qRT-PCR assay. mRNA expression increased to 7.34-fold higher at 72-h post infection compared to the control, and mRNA expression was directly correlated with the abundance of specific transgene transcripts.

The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed low virus titer compared to wild-type control plants upon challenging them with viruliferous whiteflies in a contained environment. From the results, it was concluded that amplicon V2 RNAi construct was able to limit virus replication and can be used to control CLCuV in the field.

Introduction of multiple traits in crop plants for desirable character is an essential tool of Biotechnological application. To overcome problem of insect pests and weeds cotton variety FBS-37 was genetically modified by transformation of CEMB Cry1Ac+Cry2A and GTGene cloned in different plant expression vectors under CaMV35S Promoter and nopaline synthase Nos Terminator. CEMB optimized protocol of cotton gene transformation i.e. Agrobacterium mediated shoot apex cut method was applied for transformation of these genes constructs in cotton. Overall transformation efficiency was found to be 1.05%. Putative transgenic cotton plants were shifted to soil pots and acclimatized in the green house conditions. PCR with gene specific primers amplified 190bp for EPSPS (Glyphosate GTG) and 1000bp for each of Cry1Ac and Cry2A. Quantification of Cry1Ac, Cry2A and GTG protein through ELISA determined maximum of 0.8 µ g/g, 1 µ g/g and 0.9 µ g/g of tissue respectively. 100% mortality was obtained in 2 nd instar larvae of Heliothus armigera when compared with non-transgenic control cotton plants where maximum damaged was seen in leaf bioassay. Moreover, transgenic cotton plants successfully survived when sprayed with 1600ml/acre of glyphosate as compared to control where 100% mortality and necrosis occur after 7 days of spray assay in control conditions. Keyword: Herbicide resistant, glyphosate, Cry1Ac, GTG, weedicide resistant

RNA interference is a defense response against virus multiplication. The goal of this paper was to test if expressing virus-specific shRNAs against potato virus Y (PVY) could confer resistance against PVY in potatoes. The recombinant binary vectors consisted of short hairpin RNA (shRNA) transgenes, expressed under the CaMV35S promoter, directed against the conserved portion of the potato virus Y (PVY) coat protein gene. To provide the shRNA a particular shape and stability in vivo, stem and loop sequences from a microRNA (miR403; an active regulatory microRNA in potato) were included with the siRNA sequences. The shRNA constructs were delivered into the “Cardinal” potato variety using Agrobacterium, and transgenic status was confirmed by polymerase chain reaction (PCR) and Southern blot. Compared to wild-type plants inoculated with PVY, expression of PVY coat protein mRNA, as assayed by reverse transcriptase (RT)-PCR, was reduced to 0.05% in shRNA1-transgenic potato lines and 22% in shRNA4-transgenic lines. In addition, via northern blots, the maximum accumulation of the transgene-specific siRNA was observed in shRNA1 transgenic plants, less was observed in shRNA4-transgenic lines, and the least amount was detected in control plants, which correlates with silencing of the PVY coat protein gene. Conclusively, by simultaneous RNA silencing, shRNA1-transgenic plants were found to be almost immune to PVY infection compared with shRNA4-potato plants. Therefore, the expression of virus-specific shRNAs is a novel, effective, and predictable approach to engineer resistance against PVY.

Plant growth promoting rhizobacteria are colonized bacterial species that has the capability to improve plant growth by certain direct and indirect means. Environmental factors including both biotic and abiotic stresses are among the major constraints to crop production. In the current study, the effectiveness of microbial inoculation (Bacillus megaterium) for enhancing growth of tomato plants under salt stress conditions has been investigated. Significant improvement in shoot length, root length, leaf surface area, number of leaves, total weight of the shoot and root was observed in tomato plants inoculated with zm7 strain post 15 and 30 days of its application. Zm3, Zm4 and Zm6 strains improved the morphological parameters as compared to the control. Chlorophyll content a, chlorophyll content b, anthocyanin and carotenoid content was increased in tomato plants subjected to Zm7, Zm6 and Zm4 strains. Stress responsive genes; metallothionein and glutothion gene were found highly expressed in Zm7 treated tomato plants as compared to control, untreated plants. Significant correlation of anthocyanin was reported for carotenoids, chlorophyll-b, shoot weight and total weight of seedling while carotenoids were significantly correlated with leaf surface area, root length, chlorophyll-b and anthocyanin. Overall, Zm7 strain proved best for improvement in salt stressed plant’s morphological parameters and biochemical parameters as compared to control, untreated plants.

Biological control is a novel approach in crop protection. Bacteria, such as Bacillus spp. and Pseudomonas spp., are reported for this purpose and some of their products are already commercially available. In this study, the rhizosphere and phyllosphere of healthy cotton plants were used as a source of bacterial isolates with properties of potential biocontrol agents. The isolates were screened for phosphate solubilization activity, indole acetic acid (IAA) production and antifungal activity. Two isolates, S1HL3 and S1HL4, showed phosphate solubilization and IAA production simultaneously, while another two, JS2HR4 and JS3HR2, demonstrated potential to inhibit fungal pathogens. These bacteria were identified as Pseudomonas aeruginosa (S1HL3), Burkholderia sp. (S1HL4) and Bacillus sp. (JS2HR4 and JS3HR2) based on biochemical and molecular characteristics. The isolates were tested against Cotton leaf curl virus (CLCuV) in greenhouse conditions, both as individual bacterial isolates and consortia. Treated plants were healthy as compared to control plants, where up to 74% of the plants were symptomatic for CLCuV infection. Maximum inhibition of CLCuV was observed in the plants treated with a mixture of bacterial isolates: the viral load in the treated plants was only 0.4% vs. up to 74% in controls. This treatment consortium included P. aeruginosa S1HL3, Burkholderia sp. S1HL4 and Bacillus spp. isolates, JS2HR4 and JS3HR2. The principal-component biplot showed a highly significant correlation between the viral load percentage and the disease incidence.

Graphical abstract, Expression of the transgene with a desirable character in crop plant is the ultimate goal of transgenic research. Transformation of two Bt genes namely Cry1Ac and Cry2A cloned as separate cassette under 35S promoter in pKHG4 plant expression vector was done by using shoot apex cut method of Agrobacterium. Molecular confirmation of putative transgenic cotton plants for Cry1Ac, Cry2A and GT gene was done through PCR and ELISA. Transformation efficiency of CRSP-1 and CRSP-2 was calculated to be 1.2 and 0.8% for Cry1Ac while 0.9 and 0.6% for Cry2A and 1.5 and 0.7% for GTG respectively. CRSP-1 was found to adopt natural environment (acclimatized) earlier than CRSP-2 when exposed to sunlight for one month. Expression of Cry1Ac, Cry2A and GTG was found to be 1.2, 1 and 1.3 ng/μl respectively for CRSP-1 as compared to CRSP-2 where expression was recorded to be 0.9, 0.5 and 0.9 ng/μl respectively. FISH analysis of the transgenic CRSP-1 and CRSP-2 demonstrated the presence of one and two copy numbers respectively. Similarly, the response of CRSP-1 against Glyphosate @1900 ml/acre was far better with almost negligible necrotic spot and efficient growth after spray as compared to CRSP-2 where some plants were found to have necrosis and negative control where the complete decay of plant was observed after seven days of spray assay. Similarly, almost 100% mortality of 2nd instar larvae of Heliothis armigera was recorded after three days in CRSP-1 as compared CRSP-2 where insect mortality was found to be less than 90%. Quantitatively speaking non transgenic plants were found with 23–90% leaf damage by insect, while CRSP-1 was with less than 5% and CRSP-2 with 17%. Taken together CRSP1 was found to have better insect control and weedicide resistance along with its natural ability of genetic modification and can be employed by the valuable farmers for better insect control and simultaneously for better production.

Cold-induced sweetening (CIS), which occurs when potatoes stored at low temperature for long-term, causes changes in flavor and unwanted dark colors in fried and roasted potatoes. The culprit for CIS is an enzyme called invertase. Five potato cultivars A (FD3-10), B (FD3-9), C (TPS-9801), D (393574-61) and E (Laal-e-Faisal) were investigated for invertase activity at cold storage. The correlation between invertase enzyme, sugar content and certain acids is revealed in this study. Invertase activity at 4C was up to 6.3 nmol/min/mg of protein in the cultivars A and B; 2.5 times less in cultivar C; fructose 6.4 instead of glucose in cultivar D and 3.5 times less in the cultivar E compared with same at 4C. Malic acid concentration was found to be positively correlated with invertase activity at 4C and a significant increase at low temperature storage was revealed as compared with harvesting time. However, citric acid and oxalic acid concentrations were decreased in storage. The transcript level of invertase was found significantly high in potato tubers stored at 4C in cultivar A, B and D., less in cultivar C and negligible in cultivar E. In conclusion, LAAL-E-Faisal and TPS-9801 may be used for future variety improvement programs through breeding and molecular approaches. Practical Applications Potatoes are consumed in natural as well as in processed form of foods. French fries and crisps are important subdivisions of the processing industry and denote more than 70% of the potato processing industries. Potatoes are mandatory for use throughout year, however, generating fresh potatoes all the way through the year is unrealistic and long term storage is needed. Presently cold-induced sweetening (CIS) is considered as one of the serious issue in potato production and processing with potatoes tolerant of CIS becoming a priority in many potato research programme. Potatoes resistant to CIS can deliver numerous economic benefits, such as reduction in use of sprout inhibitors, decreased dry matter loss, easy handling of high relative humidity, decreased pathogen problems during storage and reduction of chilling injury during harvest, storage and transit. Vacoular acid invertase gene is the main culprit in causing CIS. Genetic manipulation can be the best solution to generate CIS resistant potato. Most importantly, level of the carcinogenic element acrylamide can be greatly reduced by using CIS resistant varieties as it is also the byproduct of CIS. The CIS resistant potato generated through transformation can also provide valuable source for screening as well.

Plants are the primary source of nutrition and essential to maintain life on earth. They have evolved very delicate and advanced photo-sensory antennae to sense their outer environment and transduce the received information for their growth and development accordingly. This “light switch” phenomenon of plants has slowly being unraveled and various plant photoreceptors, their role in downstream molecular signaling, mutual interaction, response to circadian cycle and light signals have been discovered. The photosensory antennae in plants; phytochromes, cryptochromes and phototropins play a very crucial role in sensing the ambient light intensities. By direct interaction with the environment through these photosensory antennae, plants shift their homeostasis to regulate their growth and development. The phytochrome light receptors of plants are responsive to R/FR light and by inducing signaling pathways, trigger the physiological responses such as germination and flowering. The phytochromes also directly contribute to plant development by affecting its photosynthetic rate. To elucidate the role of phytochromes in plant metabolism, this review will focus on the importance of phytochromes, their mechanism of action and their application as an emerging field in plant biology.

BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 μg/g tissue of Cry1Ac and 0.568 μg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.

Petunia is an important and beautiful ornamental flowering plant, grown throughout the world for its beauty and attraction. Different Petunia hybrids have been developed by petunia growing countries of the world. The prescribed study was conducted to investigate the association of abscisic acid with seed yield and its contributing traits of petunia line. Data for different physiological, morphological and petunia seed yield traits was recorded, analyzed and interpreted for final inferences. From results it was showed that the petunia lines IAGS-P8, IAGS-P9 and IAGS-P11 performed well for most of the studied traits. It was shown from multivariate analysis techniques that stomata conductance, chlorophyll b contents, seed area, chlorophyll a contents, flower fresh mass, flowers per plant, seed mass and abscisic acid contributed higher to seed yield per plant in petunia. The abscisic acid contents showed positive and significant association and contribution towards seed yield of petunia genotypes. It was suggested that selection on the basis of abscisic acid may be useful to develop good seed yield per plant and large number of flowers per plant in petunia under stressful environmental conditions.

Potato virus Y has emerged as a threatening problem in all potato growing areas around the globe. PVY reduces the yield and quality of potato cultivars. During the last 30 years, significant genetic changes in PVY strains have been observed with an increased incidence associated with crop damage. In the current study, computational approaches were applied to predict Potato derived miRNA targets in the PVY genome. The PVY genome is approximately 9 thousand nucleotides, which transcribes the following 6 genes:CI, NIa, NIb-Pro, HC-Pro, CP, and VPg. A total of 343 mature miRNAs were retrieved from the miRBase database and were examined for their target sequences in PVY genes using the minimum free energy (mfe), minimum folding energy, sequence complementarity and mRNA-miRNA hybridization approaches. The identified potato miRNAs against viral mRNA targets have antiviral activities, leading to translational inhibition by mRNA cleavage and/or mRNA blockage. We found 86 miRNAs targeting the PVY genome at 151 different sites. Moreover, only 36 miRNAs potentially targeted the PVY genome at 101 loci. The CI gene of the PVY genome was targeted by 32 miRNAs followed by the complementarity of 26, 19, 18, 16, and 13 miRNAs. Most importantly, we found 5 miRNAs (miR160a-5p, miR7997b, miR166c-3p, miR399h, and miR5303d) that could target the CI, NIa, NIb-Pro, HC-Pro, CP, and VPg genes of PVY. The predicted miRNAs can be used for the development of PVY-resistant potato crops in the future.

Plants are frequently exposed to wide range of harsh environmental factors, such as drought, salinity, cold, heat, and insect attack. Being sessile in nature, plants have devel‐ oped different strategies to adapt and grow under rapidly changing environments. These strategies involve rearrangements at the molecular level starting from transcription, regu‐ lation of mRNA processing, translation, and protein modification or its turnover. Plants show stress-specific regulation of transcription that affects their transcriptome under stress conditions. The transcriptionally regulated genes have different roles under stress response. Generally, seedling and reproductive stages are more susceptible to stress. Thus, stress response studies during these growth stages reveal novel differentially regu‐ lated genes or proteins with important functions in plant stress adaptation. Exploiting the functional genomics and bioinformatics studies paved the way in understanding the rela‐ tionship between genotype and phenotype of an organism suffering from environmental stress. Future research programs can be focused on the development of transgenic plants with enhanced stress tolerance in field conditions based upon the outcome of genomic approaches and knowing the mystery of nucleotides sequences hidden in cells.

Our knowledge of the concentrations of bioaerosols in residential micro-environments in low income countries is scanty. The present investigation was conducted to assess the culturable concentration and size distribution of bacteria, gram negative bacteria and fungi in two rural and an urban site in Pakistan. The highest indoor culturable bacteria concentration was found at Rural Site II (14,650 CFU/m3) while the outdoor maximum occurred at the urban site (16,416 CFU/m3). With reference to fungi, both indoor and outdoor concentrations were considerably higher at Rural Site I than the other sites. The size distribution of culturable bacteria at all sites showed greater variability than that of culturable fungi. At all sites more than the half (55-93%) of the culturable bacterial and fungal counts were observed in the respirable fraction (

The phytochrome B (PHYB) gene of Arabidopsis thaliana was introduced into cotton through Agrobacterium tumefaciens. Integration and expression of PHYB gene in cotton plants were confirmed by molecular evidence. Messenger RNA (mRNA) expression in one of the transgenic lines, QCC11, was much higher than those of control and other transgenic lines. Transgenic cotton plants showed more than a two-fold increase in photosynthetic rate and more than a four-fold increase in transpiration rate and stomatal conductance. The increase in photosynthetic rate led to a 46% increase in relative growth rate and an 18% increase in net assimilation rate. Data recorded up to two generations, both in the greenhouse and in the field, revealed that overexpression of Arabidopsis thaliana PHYB gene in transgenic cotton plants resulted in an increase in the production of cotton by improving the cotton plant growth, with 35% more yield. Moreover, the presence of the Arabidopsis thaliana PHYB gene caused pleiotropic effects like semi-dwarfism, decrease in apical dominance, and increase in boll size.

Cormels pieces of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) formed friable calli when cultured in vitro on Murashige and Skoog basal medium containing various concentrations of auxin and cytokinin. The friable calli established cell suspensions. Plantlet regeneration was obtained from the control callus, control cell suspension derived callus and in vitro selected Fusarium oxysporum Schlecht. resistant cell-lines of Friendship. The in vitro cormlets showed 85-95% germination after breaking dormancy of 8 weeks at 4 degrees C. Cell suspensions of all four Gladiolus cultivars were found to be highly sensitive to fusaric acid. Gradual increase in fusaric acid concentrations to the cell-suspension cultures decreased cell growth considerably. One albino plant was found from the second generation of the in vitro selected cell line of Friendship. The albino plant was found to be highly susceptible to F. oxysporum. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of the F. oxysporum before planting and were also sprayed with the same spore suspension for further characterization when the height of plants was about 6 cm. The four selected cell lines showed the same response whether or not they were inoculated with conidia of the F. oxysporum. Plantlets of all of the selected cell lines exhibited significant growth as compared with the control after application of conidia of the F. oxysporum.

Fusarium oxysporum is an important pathogen that can reduce corm and flower production of Gladiolus. Cell suspensions of four Fusarium susceptible Gladiolus cultivars (Friendship, Peter Pears, Victor Borge and Novalux) were found highly sensitive to the fusaric acid (FA). Gradual increase in FA concentrations to the cell-suspension cultures decreased cell growth considerably. Plantlets of all the selected cell lines showed significant resistance as compared with control in the presence of 0.5 mM FA. The in vitro selected cell line numbers CAMB-G01, CAMB-G04, CAMB-G06 and CAMB-G09 did not affect at all and showed an Average Severity Index of zero as compared with the control of the Friendship cultivar. The cormlets of all in vitro selected cell lines of Friendship were inoculated with a conidial suspension of Fusarium oxysporum before planting and also sprayed with the same spore suspension when the height of plants was about 6 cm for further characterization. The four selected cell lines showed the same response whether or not inoculated with conidia of Fusarium oxysporum. Plantlets of all the selected cell lines exhibited significant growth as compared with control after application of conidia of Fusarium oxysporum.

More than 50 countries around the globe cultivate cotton on a large scale. It is a major cash crop of Pakistan and is considered “white gold” because it is highly important to the economy of Pakistan. In addition to its importance, cotton cultivation faces several problems, such as insect pests, weeds, and viruses. In the past, insects have been controlled by insecticides, but this method caused a severe loss to the economy. However, conventional breeding methods have provided considerable breakthroughs in the improvement of cotton, but it also has several limitations. In comparison with conventional methods, biotechnology has the potential to create genetically modified plants that are environmentally safe and economically viable. In this study, a local cotton variety VH 289 was transformed with two Bt genes (Cry1Ac and Cry2A) and a herbicide resistant gene (cp4 EPSPS) using the Agrobacterium mediated transformation method. The constitutive CaMV 35S promoter was attached to the genes taken from Bacillus thuringiensis (Bt) and to an herbicide resistant gene during cloning, and this promoter was used for the expression of the genes in cotton plants. This construct was used to develop the Glyphosate Tolerance Gene (GTGene) for herbicide tolerance and insecticidal gene (Cry1Ac and Cry2A) for insect tolerance in the cotton variety VH 289. The transgenic cotton variety performed 85% better compared with the non-transgenic variety. The study results suggest that farmers should use the transgenic cotton variety for general cultivation to improve the production of cotton.

Background Cotton yield has been badly affected by different insects and weed competition. In Past Application of multiple chemicals is required to manage insects and weed control was achieved by different conventional means, such as hand weeding, crop rotation and polyculture, because no synthetic chemicals were available. The control methods shifted towards high input and target-oriented methods after the discovery of synthetic herbicide in the 1930s. To utilise the transgenic approach, cotton plants expressing the codon-optimised CEMB GTGene were produced in the present study. Results Local cotton variety CEMB-02 containing Cry1Ac and Cry2A in single cassette was transformed by synthetic codon-optimised 5-enolpyruvylshikimate-3-phosphate synthase gene cloned into pCAMBIA 1301 vector under 35S promoter with Agrobacterium tumifaciens. Putative transgenic plants were screened in MS medium containing 120 µmol/L glyphosate. Integration and expression of the gene were evaluated by PCR from genomic DNA and ELISA from protein. A 1.4-kb PCR product for Glyphosate and 167-bp product for Cry2A were obtained by amplification through gene specific primers. Expression level of Glyphosate and Bt proteins in two transgenic lines were recorded to be 0.362, 0.325 µg/g leaf and 0.390, 0.300 µg/g leaf respectively. FISH analysis of transgenic lines demonstrates the presence of one and two copy no. of Cp4 EPSPS transgene respectively. Efficacy of the transgene Cp4 EPSPS was further evaluated by Glyphosate spray (41 %) assay at 1900 ml/acre and insect bioassay which shows 100 %mortality of insect feeding on transgenic lines as compared to control. Conclusion The present study shows that the transgenic lines produced in this study were resistant not only to insects but also equally good against 1900 ml/acre field spray concentration of glyphosate. Electronic supplementary material The online version of this article (doi:10.1186/s13104-015-1397-0) contains supplementary material, which is available to authorized users.

Unnecessary weed growth in sugarcane fields forces plants to compete for nutrients and sunlight for survival, which most often leads to significant yield losses. As chemical herbicides cannot differentiate between crop plants and weeds, the development of herbicide-tolerant crops is anticipated. Four sugarcane varieties, CPF-234, CPF-213, HSF-240, and CPF-246, were used to develop glyphosate herbicide tolerance. A glyphosate-tolerant gene of 1368 bp cloned directionally under the 35S promoter with the \Beta-glucuronidase (GUS) reporter gene was used as the transgene. Through the biolistic transformation of sugarcane, calli of all cultivars were transformed with glyphosate-tolerant gene constructs. Efficient regeneration conditions were optimized on 1.5-2.5 mg/L kinetin, 1.5-2.5 mg/L 6-benzylaminopurine (BAP), and 1 mg/L gibberellic acid (GA3). The transformants exhibited the best shoot regeneration on a medium containing 2 mg/L kinetin, 2 mg/L BAP, 2 mg/L GA3, and 1 mg/L indole-3-acetic acid. Based on initial screenings through GUS assay, the transformation efficiency was 22%, 32%, 17%, and 13% for cultivars 246, 234, 213, and 240, respectively. In transformed sugarcane plants, the transgene of 1368 bp was amplified. ELISA with gene-specific coated monoclonal IgG confirmed the transgene protein expression. It was revealed that all acclimatized transgenic sugarcane plants survived the glyphosate spray application of 900 mL/0.404 ha, except for the control nontransformed plants. However, at spray application of 1100 mL/0.404 ha, transgenic plants having the transgene protein OD of 0.2 to 1.0 did not survive, while those that had a transgene protein OD range of between 1.0 and 2.0 did. In addition, weeds growing alongside transgenic sugarcane plants turned brown and subsequently died at glyphosate spray applications of both 900 and 1100 mL/0.404 ha.

Sugarcane is an important sugar and cash crop grown throughout the world. Sugarcane quality and production is mostly affected by biotic and abiotic factors that caused low yield of sugarcane products throughout the world. Using biotechnology and genetic engineering as a complement for traditional breeding methods it is possible to introduce insect/pest, herbicide-tolerant traits into various crop species. It has been found that the absence of insect/pest, abiotic and herbicide tolerant genes in genetic pool of crop plant species makes traditional breeding programs difficult. The varieties that are productive and at the same time has resistance against certain pathogens and diseases, can improve yield. The development of biotic and abiotic resistant sugarcane varieties through transgenic technology will be cost effective in controlling all type of stresses which ultimately improve yield potential. The present review will provide its readers the opportunity to understand the methods of glyphosate tolerant and Bt resistant genes in sugarcane.

RNA mediated silencing technology has now become the tool of choice for induction of virus resistance in plants. A significant feature of this technology is the presence of doublestranded RNA (dsRNA), which is not only the product of RNA silencing but also the potent triggers of RNA interference (RNAi). Upon RNAi induction, these dsRNAs are diced into short RNA fragments termed as small interfering RNAs (siRNAs), which are hallmarks of RNAi. Considerable resistance in transgenic plants against viruses can be created by exploiting the phenomenon of RNAi. In the current chapter, generation of potato virus Y (PVY) resistant potato and sugarcane mosaic virus (SCMV) resistant sugarcane by CEMB has been quoted as an example.