Engineering of insecticidal hybrid gene into potato chloroplast genome exhibits promising control of Colorado potato beetle, Leptinotarsa decemlineata (Coleoptera: Chrysomelidae).

The potato chloroplast was transformed with codon optimized synthetic hybrid cry gene (SN19) to mitigate crop losses by Colorado potato beetle (CPB). The bombarded explants (leaves and internode) were cultured on MS medium supplemented with BAP (2.0 mg/l), NAA (0.2 mg/l), TDZ (2.0 mg/l) and GA3 (0.1 mg/l); spectinomycin 50 mg/l was used as a selection agent in the medium. Leaf explants of cultivar Kuroda induced highest percentage (92%) of callus where cultivar Santae produced the highest percentage (85.7%) of transplastomic shoots. Sante and Challenger showed 9.6% shoot regeneration efficiency followed by cultivar Simply Red (8.8%). PCR amplification yielded 16 postive transplastomic plantlets out of 21 spectinomycin resistant ones. Target gene integration was confirmed by PCR and Southern blot, whereas RT-qPCR was used to assess the expression level of transgene. The localization of visual marker gene gfp was tracked by laser scanning confocal microscopy which confirmed its expression in chloroplasts of leaf cells. The transplastomic plants ensured high mortality to both larvae and adult CPB. Foliage consumption and weight gain of CPB fed on transplastomic leaves were lower compared to the control plants. Sucessful implementation of current research findings can lead to a viable solution to CPB mediated potato losses globally.

Modern Trends in Plant Genome Editing: An Inclusive Review of the CRISPR/Cas9 Toolbox.

Increasing agricultural productivity via modern breeding strategies is of prime interest to attain global food security. An array of biotic and abiotic stressors affect productivity as well as the quality of crop plants, and it is a primary need to develop crops with improved adaptability, high productivity, and resilience against these biotic/abiotic stressors. Conventional approaches to genetic engineering involve tedious procedures. State-of-the-art OMICS approaches reinforced with next-generation sequencing and the latest developments in genome editing tools have paved the way for targeted mutagenesis, opening new horizons for precise genome engineering. Various genome editing tools such as transcription activator-like effector nucleases (TALENs), zinc-finger nucleases (ZFNs), and meganucleases (MNs) have enabled plant scientists to manipulate desired genes in crop plants. However, these approaches are expensive and laborious involving complex procedures for successful editing. Conversely, CRISPR/Cas9 is an entrancing, easy-to-design, cost-effective, and versatile tool for precise and efficient plant genome editing. In recent years, the CRISPR/Cas9 system has emerged as a powerful tool for targeted mutagenesis, including single base substitution, multiplex gene editing, gene knockouts, and regulation of gene transcription in plants. Thus, CRISPR/Cas9-based genome editing has demonstrated great potential for crop improvement but regulation of genome-edited crops is still in its infancy. Here, we extensively reviewed the availability of CRISPR/Cas9 genome editing tools for plant biotechnologists to target desired genes and its vast applications in crop breeding research.

Histopathological changes induced in liver, kidney, heart and pancreas of rabbits by prolonged oral cyanide exposure.

The aim of the present study was to determine the deleterious effects of prolonged oral cyanide insult on various organs and tissues in rabbits. For this purpose, 12 locally bred adult male rabbits were allocated into two groups of 6 viz. control and experimental. Rabbits in control group were offered feed only while the rabbits in experimental group received feed plus potassium cyanide (KCN) at 3 mg/kg body weight orally for a period of 40 days. None of the rabbit in both the groups demonstrated any of the gross changes in any organ on postmortem examination. Liver was normal in size, shape, texture and color. Kidneys were also normal in size and color. Histopathological examination revealed severe hepatocyte vacuolation and degeneration in liver of rabbits in experimental group. There was also excessive congestion in liver and bile duct of rabbits in experimental group. Kidneys of rabbits in experimental group demonstrated severe glomerular and tubular necrosis and congestion. In the tubular epithelial cells, pyknotic nuclei were also present. On the other hand, heart and pancreas of rabbits in both control and experimental group did not show any histopathological change in microscopic structures. In conclusion, prolonged oral cyanide administration could have harmful effects on liver and kidney functions.

Assessing the efficacy of aqueous garlic extract, sodium nitrite and sodium thiosulfate against prolonged oral cyanide exposure in rabbits.

This study was aimed to compare the efficacy of aqueous garlic extract, sodium nitrite (SNT), sodium thiosulfate (STS) and hydroxocobalamin against oral cyanide exposure in rabbits. For this purpose, forty two adult male rabbits were divided randomly into 7 groups of 6 animals (A-G) each. Rabbits in group A were offered feed only and served as negative control, while the rabbits in group B received feed plus potassium cyanide (KCN) at 3mg/kg orally and were kept as positive control. Animals in group C received feed, KCN and intraperitoneal injection (IP) of aqueous garlic extract at 500mg/kg. Rabbits in group D were given feed, KCN and IP injection of STS at 600mg/kg. Members in group E received feed, KCN and IP injection of both aqueous garlic extract at 500mg/kg and SNT at 20mg/kg. Animals in group F were given feed, KCN and IP injection of both STS at 600mg/kg and SNT at 20mg/kg, while the rabbits in group G received feed, KCN and IP injection of hydroxocobalamin at 300mg/kg. The treatments were given to respective groups for 40 days. The efficacy of the antidotes was measured on the basis of changes in biochemical profile of rabbits in each group. In this study, hydroxocobalamin was found to be significantly more effective cyanide (CNI) antidote than garlic, STS, SNT plus garlic extract, or SNT and STS, either alone or in combination. A combination of SNT and garlic extract was the second most effective CNI antidote. The efficacy of garlic alone was significantly higher than STS alone or in combination with SNT. The efficacy of combined SNT and STS was superior to STS alone in treating rabbits with CNI toxicity. In conclusion, aqueous garlic extract alone or in combination with STS can effectively be used against cyanide toxicity.

Risk factors associated with Mycobacterium bovis skin positivity in cattle and buffalo in Peshawar, Pakistan.

A cross-sectional study was carried out to determine the prevalence of bovine tuberculosis (bTB) and associated risk factors in cattle and buffalo in Peshawar, Pakistan. Cattle and buffalo, randomly selected from all four towns of District Peshawar, were screened for bovine tuberculosis using comparative cervical intradermal tuberculin test (CCIT). For obtaining data on risk factors, sociodemographic condition, animal characteristics, and management, interviewer administered pretested questionnaire to animal owners. Multivariable logistic regression models were used to measure association between risk factors and comparative cervical intradermal tuberculin reactors. A total of 556 cattle and buffalo were screened for bovine tuberculosis. Out of 556 animals screened, 5.75 % (3.9-8.0 %) were found positive. The prevalence was higher in old animals (p = 0.001) as compared to younger animals. Prevalence also varied with source of animal (either raised on farm or purchased), stay of animals at night (indoor or outdoor), and herd size. Farmer's knowledge about transmission of TB from animals to human as well as signs and symptoms of TB was extremely low. Only 3.6 % farmers correctly stated the combination of three major symptoms of TB. Results of the study call for immediate intervention to control bTB in animals as well as its transmission to human population. Furthermore, it is suggested to emphasize on local epidemiology of bTB and husbandry practices of cattle and buffalo during the control program.

Prolonged oral cyanide effects on feed intake, growth rate and blood parameters in rabbits.

Twelve adult rabbits bred locally were divided into two equal groups of 6; experimental and control groups. Rabbits in the experimental group were orally dosed with KCN at 3mg/kg body weight for 40 consecutive days. Members in control group were given placebo (distilled water) for the same period. Animals in both groups were offered feed at 90gm/kg/day while ample drinking water was available ad lib. Feed consumption and body weight of rabbits in both the groups were recorded. Blood samples were also drawn to determine various hematological parameters. Statistical analysis revealed a non-significant difference of total and daily feed intakes in rabbits of experimental and control groups. Whereas the feed efficiency of rabbits in the experimental group were significantly reduced (P<0.05) compared to controls. Likewise a significant decrease in body weight gain of rabbits in experimental group (P<0.05) was observed. A non-significant difference (P>0.05) was observed in leukocyte count, differential leukocyte count and platelets of rabbits in both the groups. Erythrocyte count, hemoglobin concentration, packed cell volume and mean corpuscular hemoglobin were significantly decreased in treated rabbits. It was concluded that chronic cyanide intake had a deleterious effect on feed efficiency, growth rate and blood components of rabbits.

Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant.

The production of interferon alpha from microbial to mammalian expression system, have certain precincts in terms of cost, scalability, safety and authenticity. Modern biotechnology exploits transgenic crops to get large quantities of complex proteins in a cost-effective way. In order to overcome several challenges from biosafety point of view, the chloroplast transformation strategy is one of the best approaches since plastids are strictly maternally inherited in most of the cultivated species. In the present study the interferon alpha 5 gene was synthesized by using complex set of oligos. After sequence confirmation of the synthesized gene, the histidine residues along with the thrombin protease site were engineered upstream to the synthetic interferon alpha 5 gene. The recombinant fragment was then tethered with chloroplast light inducible promoter, rbcl followed by sequential cloning to develop chloroplast transformation vector to target the cassette into the inverted repeat region of plastome through two events of homologous recombination. The putative transgenic plants obtained through biolistic delivery method and as a result of antibiotic selection of bombarded leaves, were subjected to different rounds of selection and regeneration for homoplasmicity. The spectinomycin-resistant shoots were analyzed through Polymerase Chain Reaction and Sothern blotting. The expression of introduced synthetic genes was recorded using Enzyme Linked ImmunoSorbant Assay technique. It was experienced that mature leaves contained comparatively high levels of interferon compared to young and senescence leaves.

Chloroplast-encoded chlB gene from Pinus thunbergii promotes root and early chlorophyll pigment development in Nicotiana tabaccum.

Chlorophyll biosynthesis is catalyzed by two multi subunit enzymes; a light-dependent and a light-independent protochlorophyllide oxidoreductase. The light-independent enzyme consists of three subunits (ChlL, ChlN and ChlB) in photosynthetic bacteria and plastids in which the chlB gene encodes the major subunit that catalyzes the reduction of protochlorophyllide to chlorophyllide. We report here stable integration of the chlB gene from Pinus thunbergii into the chloroplast genome of tobacco. Using helium-driven biolistic gun, transplastomic clones were developed in vitro. The stable integration and homoplasmy for transgenes was confirmed by using PCR and Southern blotting techniques. Nodal cuttings of the homoplasmic transgenic and untransformed wild type shoots were cultured on MS medium in the dark. As expected, shoots developed from the cuttings of the wild type plants in the dark showed etiolated growth with no roots whereas shoots from the cuttings of the transgenic plants developed early and more roots. Upon shifting from dark to light in growth room, leaves of the transgenic shoots showed early development of chlorophyll pigments compared to the wild type shoots. Further, photosynthetically indistinguishable transgenic shoots also showed significant difference in root development from untransformed wild type shoots when cuttings were grown in the light. Therefore, it may be concluded that the chlB gene is involved, directly or indirectly, in the root development of tobacco. Further, the gene promotes early development of chlorophyll pigments, upon illumination from dark, in addition to its role in the light-independent chlorophyll formation when expressed together with subunits L&N in other organisms.

The tobacco chloroplast YCF4 gene is essential for transcriptional gene regulation and plants photoautotrophic growth.

A tobacco chloroplast hypothetical open reading frame 4 (YCF4) has been reported as a non-essential assembly factor for photosynthesis based on an incomplete knockout of YCF4, just 93 of 184 amino acids from the N-terminus were knocked out. On the other hand, we removed the complete sequence of YCF4 from tobacco chloroplasts and observed that ΔYCF4 plants were unable to survive photoautotrophically as their growth was hampered in the absence of an external carbon supply, clearly showing that the YCF4 is essential for photosynthesis. Initially, the aadA gene was introduced into the tobacco plastome replacing the complete YCF4 gene through homologous recombination events. The replacement of YCF4 with aadA was confirmed by PCR and Southern blot analysis in ΔYCF4 plants. Homoplasmic ΔYCF4 plants had a light green phenotype, and the leaves became pale yellow as the plants grew older. The structure of chloroplasts of ΔYCF4 mutants of light green phenotype was studied using a transmission electron microscope (TEM), and the micrographs demonstrated structural anomalies in the chloroplasts; including shape, size, and grana stacking compared to the wild-type plants. Further, transcriptome analysis revealed that the expression of PSI, PSII, and ribosomal genes remained unchanged in ∆YCF4 plants. On the other hand, transcriptome levels of rbcL (Ribulose 1,5-bisphosphate carboxylase/oxygenase large subunit), LHC (Light-Harvesting Complex), and ATP Synthase (atpB and atpL) decreased, indicating that the YCF4 has the function(s) in addition to assembling the photosynthetic complex. This was confirmed by in-silico protein-protein interactions of full-length YCF4 as well as 93 and 91 of 184 amino acids from N- and C-termini of the full-length protein, which revealed that the C-terminus (91 aa) of YCF4 is important in interacting with other chloroplast proteins. These findings provide genetic support for the plastid YCF4 gene's critical role in regulating the plastid gene expression and assembling the photosynthetic complex.

Developmental and photosynthetic regulation of δ-endotoxin reveals that engineered sugarcane conferring resistance to 'dead heart' contains no toxins in cane juice.

The phosphoenolpyruvate (PEP) carboxylase is regulated at the levels of transcription and post-translation in C4 plants in light and abundantly accumulates in leaf mesophyll cells. We report here developmental and photosynthetic regulation of stably accumulated Bacillus thuringiensis δ-endotoxin under the control of PEP-C promoter in transgenic sugarcane. In young leaves of plants, the transprotein is accumulated to 39% of the levels in mature leaves (135 ng mg(-1)), and is induced with the cell development, from base to tip. Nevertheless, these levels are decreased up to 99.98% in non-photosynthetic cells as cane matures, from top to bottom, suggesting the photosynthesis regulation of δ-endotoxin in cane cells. Further, transgenic plants are highly resistant to 'dead heart'. In these studies, Scirpophaga nivela larvae causing 'dead heart' were killed within one hour of release to the transgenic plants. Therefore, this report may be regarded as the first report that provides a better strategy for developing transgenic sugarcane lines with absolute protection against invading larvae and no toxin residues in cane juice.

Relationship of age to body weight, scrotal circumference, testicular ultrasonograms, and semen quality in Sahiwal bulls.

The objective of the present study was to determine the relationship of age to body weight (BW), scrotal circumference (SC), number of pixels of testicular ultrasonograms (NP), and semen quality in Sahiwal bulls. The study was based on 128 Sahiwal bulls of different age groups (from 0 to >100 months of age). Bulls were evaluated for SC, BW, and NP. Semen was evaluated once a week for five consecutive weeks from regularly collected donor bulls (n = 86) ranging in age from 25-30 to >100 months. Ejaculate volume, sperm motility, sperm concentration, sperm morphology, percent live sperms, sperm plasma membrane integrity, and normal acrosome were compared among different age groups. Mean SC and BW increased (P < 0.05) in a curvilinear manner from birth to >100 months of age. Mean NP of testicular ultrasonograms increased (P < 0.05) from 0 to 24 months and then plateaued until >100 months of age. Body weight, SC, and NP were positively correlated with age from birth until >100 months (r = 0.91, 0.87, and 0.40, respectively). Ejaculate volume (5.7 ± 0.2 vs. 4.6 ± 0.09 ml) and sperm concentration (1,281.6 ± 17.7 vs. 1,115.8 ± 55.9 × 10(6)/ml) increased (P < 0.05) in mature bulls compared to younger ones. However, motility (68.6 ± 0.3%), plasma membrane integrity (50.8 ± 1.0%), and normal acrosome (74.8 ± 0.5%) remained insignificant due to age. In six of eight age groups studied, morphological abnormalities were well within the range (18.1 ± 0.3%). In conclusion, the BW, SC, and NP of testicular ultrasonograms, ejaculate volume, and concentration increased with age. Moreover, semen quality is fairly independent of age except volume and concentration in Sahiwal bulls.

Modified picrate method for determination of cyanide in blood.

Cyanide (CN-) is widely distributed in the ecosystem and has been associated with toxic effects in humans and animals. Most outbreaks of CNÖ¿ poisoning in animals result from ingestion of plants containing cyanogenic glycosides. Various analytical techniques for estimating cyanide in blood are available. A simple picrate method was developed to determine blood CN- in goats. This assay is a modification of commonly available methods using picrate paper and those using Conway diffusion cells. Cyanide in blood was measured during and after IV administration of KCN at 0.6 mg/min for 1 h. Blood CN- levels in rabbits were determined after oral administration of KCN for 10, 20, 30 and 40 days. The CN- concentration in blood of goats was time-dependent and continued rising during infusion followed by gradual decline after infusion stopped. A calibration curve set by dissolving various concentrations of KCN in distilled water showed a linear relationship between CN- concentration and absorbance (R=0.995) ranging from 0.3-120 mg CN-/L. Blood CN- levels in rabbits showed time-dependent increase with maximum concentration (1.34 mg/L) at 40 days. This is a simple and inexpensive tool for the determination of blood CN- in the laboratory and under field conditions as well.

Immunogenic profiling and designing of a novel vaccine from capsid proteins of FMDV serotype Asia-1 through reverse vaccinology.

Foot-and-mouth disease is one of the devastating transboundary animal diseases causing heavy losses to the livestock industry. Different vaccines based on the inactivated FMD virus are used against this disease, but lack of immunological memory and the need for high biocontainment are the major drawbacks of these vaccines. A novel vaccine comprising recombinant antigenic regions is effective, as they lack viruses for production. Considering the fact, capsid proteins vp4, vp2, vp3, and vp1 with 3C protease of FMDV serotype Asia-1 were analyzed through reverse vaccinology approaches in this study. The sequence and structural analysis of the proteins is carried out through various bioinformatic tools and the sequence analysis has figured out the acidic nature and thermal stability of the proteins, likewise, the phylogenetic analysis helped us to trace the FMDV isolates, elucidating that selected proteins belong to the strain (Group VII), which is currently circulating in Pakistan. Next, the B-cell and MHC Class-I epitopes are identified from the antigenic proteins by immunoinformatic tools. The highly conserved, antigenic, and non-allergenic epitopes are used to design the vaccine. Accordingly, the codon adaptation and in silico cloning of the corresponding genes is performed. Thus, the bacterial expression vector could be used for efficient expression and large-scale production of the vaccine.

Metabolomics: A Powerful Tool to Study the Complexity of Wheat Metabolome.

Wheat is a widely cultivated cereal, consumed by nearly 80% of the total population in the world. Although wheat is growing on 215 million hectares annually, its production is still inadequate to meet the future demand of feeding the 10 billion human population. Global food security is the biggest challenge as climate change is threatening crop production. There is a need to fast-- track the wheat breeding by devising modern biotechnological tools. Climate-smart wheat having greater stress resilience, better adaptability and improved agronomic traits are vital to guarantee food security. Substantial understanding and knowledge of vital biochemical pathways and regulatory networks is required for achieving stress resilience in wheat. Metabolomics has emerged as a fascinating technology to speed up the crop improvement programs by deciphering unique metabolic pathways for abiotic/biotic stress tolerance. State-of-the-art metabolomics tools such as nuclear magnetic resonance (NMR) and advanced mass spectrometry (MS) has opened new horizons for detailed analysis of wheat metabolome. The identification of unique metabolic pathways offers various types of stress tolerance and helps to screen the elite wheat cultivars. In this review, we summarize the applications of metabolomics to probe the stress-responsive metabolites and stress-inducive regulatory pathways that govern abiotic/biotic stress tolerance in wheat and highlight the significance of metabolic profiling to characterize wheat agronomics traits. Furthermore, we also describe the potential of metabolomics-assisted speed breeding for wheat improvement and propose future directions.

Over-Expression of Endogenous SUGARWIN Genes Exalted Tolerance against Colletotrichum Infection in Sugarcane.

Sugarcane being the major contributor of sugar and potential source of biofuel around the globe, occupies significant commercial importance. Red rot is the most devastating disease of sugarcane, severely affecting its quality as well as yield. Here we report the overexpression of SUGARWIN1 and SUGARWIN2 genes in any field crop for the first time. For this purpose, SUGAWIN1 and SUGARWIN2 were cloned downstream of maize ubiquitin (Ubi-1) promoter to construct two independent expression cassettes. The bar gene conferring resistance against phosphinothricin was used as selectable marker. Embryogenic calli of sugarcane were bombarded with both expression cassettes and selected on regeneration medium supplemented with phosphinothricin. The phosphinothricin-resistant shoots were rooted and then, analyzed using molecular tools at the genomic as well as transcriptomic levels. The transcriptomic analysis, using real time qPCR, showed that expression of SUGARWIN1 (SWO) and SUGARWIN2 (SWT) was higher in transgenic plants as compared to untransformed plants. Our results further demonstrated that over expression of these genes under maize ubiquitin (Ubi-1) promoter causes significant restriction in proliferation of red rot causal agent, Colletotrichum falcatum in sugarcane transgenic plants, under in vitro conditions. This report may open up exciting possibilities to extend this technology to other monocots for the development of crops with better ability to withstand fungal pathogens.

Characterization and Expression Analysis of Resistance Gene Analogues in Elite Sugarcane Genotypes.

BACKGROUND: Resistance Gene Analogues (RGAs) are an important source of disease resistance in crop plants and have been extensively studied for their identification, tagging and mapping of Quantitative Trait Loci (QTLs). Tracking these RGAs in sugarcane can be of great help for the selection and screening of disease resistant clones. OBJECTIVE: In the present study expression of different Resistance Gene Analogues (RGAs) was assessed in indigenous elite sugarcane genotypes which include resistant, highly resistant, susceptible and highly susceptible to disease infestation. METHODS: Total cellular DNA and RNA were isolated from fourteen indigenous elite sugarcane genotypes. PCR, semi-quantitative RT PCR and real time qPCR analyses were performed. The resultant amplicons were sequence characterized, chromosomal localization and phylogenetic analysis were performed. RESULTS: All of the 15 RGA primers resulted in amplification of single or multiple fragments from genomic DNA whereas only five RGA primers resulted in amplification from cDNA. Sequence characterization of amplified fragments revealed 86-99% similarity with disease resistance proteins indicating their potential role in disease resistance response. Phylogenetic analysis also validated these findings. Further, expression of RGA-012, RGA-087, RGA-118, RGA-533 and RGA-542 appeared to be upregulated and down regulated in disease resistant and susceptible genotypes, respectively, after inoculation with Colletotrichum falcatum. CONCLUSION: RGAs are present in most of our indigenous genotypes. Anyhow, differential expression of five RGAs indicated that they have some critical role in disease resistance. So, the retrieved results can not only be employed to devise molecular markers for the screening of disease resistant genotypes but can also be used to develop disease resistant plants through transgenic technology.

Comprehensive Mechanism of Gene Silencing and Its Role in Plant Growth and Development.

Gene silencing is a negative feedback mechanism that regulates gene expression to define cell fate and also regulates metabolism and gene expression throughout the life of an organism. In plants, gene silencing occurs via transcriptional gene silencing (TGS) and post-transcriptional gene silencing (PTGS). TGS obscures transcription via the methylation of 5' untranslated region (5'UTR), whereas PTGS causes the methylation of a coding region to result in transcript degradation. In this review, we summarized the history and molecular mechanisms of gene silencing and underlined its specific role in plant growth and crop production.

Codon optimization of cry1Ab gene for hyper expression in plant organelles.

With the advent of genetic manipulation techniques, it has become possible to clone and insert gene into the genome of crop plants to confer resistance to insects and pests. Resistance to insects has been demonstrating in transgenic plants either by triggering defense system of plants or by expressing heterologous cry genes for delta-endotoxins from Bacillus thuringiensis. In the present study, synthetic cry1Ab gene was developed with optimized chloroplast preferred codons and is expressed in tobacco plastid genome called plastome, following chloroplast transformation strategy, which is environment friendly technique to minimize out-crossing of transgenes to related weeds and crops. In addition, due to high polyploidy of plastid genome transformation of chloroplast permits the introduction of thousands of copies of foreign genes per plant cell, leading to extraordinarily high levels of foreign protein expression. The chloroplast transformation technology aims to insert stably into the plastome through homologous recombination into pre-decided position. To characterize the synthetic cry1Ab gene, chloroplast transformation vectors were developed and bombarded to the leaf cells of tobacco plants maintained under aseptic conditions. After bombardment, the drug resistant shoots were selected and regenerated on drug containing regeneration medium. Homoplasmic shoots were recovered after successive rounds of selection and regeneration. Proliferated plants were subjected to genomic DNA analysis by using polymerase chain reaction (PCR) technique where cry1Ab gene-specific primers were used. PCR positive plants were subjected to protein analysis, and functionally expressed proteins were detected using Immuno-Strips specific for cry1Ab/Ac gene products. Transgenic plants carrying cry1Ab gene were found expressing Bt toxins confirming that engineered gene could be expressed in other plants as well.

Transmission of Engineered Plastids in Sugarcane, a C4 Monocotyledonous Plant, Reveals that Sorting of Preprogrammed Progenitor Cells Produce Heteroplasmy.

We report here plastid transformation in sugarcane using biolistic transformation and embryogenesis-based regeneration approaches. Somatic embryos were developed from unfurled leaf sections, containing preprogrammed progenitor cells, to recover transformation events on antibiotic-containing regeneration medium. After developing a proficient regeneration system, the FLARE-S (fluorescent antibiotic resistance enzyme, spectinomycin and streptomycin) expression cassette that carries species-specific homologous sequence tails was used to transform plastids and track gene transmission and expression in sugarcane. Plants regenerated from streptomycin-resistant and genetically confirmed shoots were subjected to visual detection of the fluorescent enzyme using a fluorescent stereomicroscope, after genetic confirmation. The resultant heteroplasmic shoots remained to segregate on streptomycin-containing MS medium, referring to the unique pattern of division and sorting of cells in C4 monocotyledonous compared to C3 monocotyledonous and dicotyledonous plants since in sugarcane bundle sheath and mesophyll, cells are distinct and sort independently after division. Hence, the transformation of either mesophyll or bundle sheath cells will develop heteroplasmic transgenic plants, suggesting the transformation of both types of cells. Whilst developed transgenic sugarcane plants are heteroplasmic, and selection-based regeneration protocol envisaging the role of division and sorting of cells in the purification of transplastomic demands further improvement, the study has established many parameters that may open up exciting possibilities to express genes of agricultural or pharmaceutical importance in sugarcane.

Seeds as Economical Production Platform for Recombinant Proteins.

The cost-effective production of high-quality and biologically active recombinant molecules especially proteins is extremely desirable. Seed-based recombinant protein production platforms are considered as superior choice owing to lack of human/animal pathogenic organisms, lack of cold chain requirements for transportation and long-term storage, easy scalability and development of edible biopharmaceuticals in plants with objective to be used in purified or partially processed form is desirable. This review article summarizes the exceptional features of seed-based biopharming and highlights the needs of exploiting it for commercial purposes. Plant seeds offer a perfect production platform for high-value molecules of industrial as well as therapeutic nature owing to lower water contents, high protein storage capacity, weak protease activity and long-term storage ability at ambient temperature. Exploiting extraordinarily high protein accumulation potential, vaccine antigens, antibodies and other therapeutic proteins can be stored without effecting their stability and functionality up to years in seeds. Moreover, ability of direct oral consumption and post-harvest stabilizing effect of seeds offer unique feature of oral delivery of pharmaceutical proteins and vaccine antigens for immunization and disease treatment through mucosal as well as oral route.