Abiotic stress like salinity is a major factor that significantly limits crop growth and productivity. It has been shown that various salt stress responsive genes participate in salt stress tolerance. Generation of transgenic plants with genes resistance to salt stress can greatly improve plant salinity tolerance. Plant specific kinases and transcription factors (TFs) play an important role in this regard. Genetic engineering of agriculturally important crops such as tomato for salt tolerance holds promising application. Local cultivar tomato ex-plant will be used for transformation of tomato plant with salt stress resistance gene. Once transformed with salt tolerance gene, tomato ex-plants will be put on different combination of rooting and shooting hormone concentration for tomato ex-plant growth. This will result in tomato plant growth and regeneration. The regenerated plantlets will be tested for presence of salt stress resistant gene and stress tolerance. The regenerated plantlets will be transferred to the green house to be planted in soil. This work will form the basis for transformation and development of transgenic salt stress resistance lines in local tomato variety. This study can be used for improved abiotic stress trait in tomato crop that will greatly benefit the agriculture industry of Pakistan

Pakistan exports tomato, an important vegetable crop, to different areas of the world and generates valuable revenue. But per hector yield is low compared to other countries. One reason is the inability of tomato varieties to cope with the stresses plant face in the field. Drought is an important stress. Plant breeders, over last fifty years, have used the available genetic diversity to develop improved varieties but have ignored epigenetic diversity. Recently, trangenerational nature of epigenetic modifications especially DNA methylation have been reported meaning that the induced change in the DNA methylation pattern can be transmitted to next generations. Therefore, in the proposed study, the shift in the DNA methylation pattern will be chemically induced and then the plants will be screened for the phenotypes of agronomic interest under drought stress. These plants will be studied phenotypically as well as epi-genotypically to identify the regions where these epi-mutations have occurred and then their transgenerational transmission will be evaluated. The plants with the stable transgenerational epi-mutations will be recommended to the breeders as well as for commercial use subjected to field trials.

Industrial waste is a major environmental problem in Pakistan and worldwide. There are different kinds of industrial waste which cause the pollution and may leads hazardous effects on the environment, health, agriculture, marine life and various others. Clean technology is an idea to recover and reuse the wastefor other purposes providing added value and many advantages. Taking this idea the industrial waste can be converted into highly valuable products including bio-ethanol many others. Recently, our group had discovered that some industrial wastes can be use as growth media for bacteria to produce bacterial cellulose (BC). BC is a biopolymer produced by a class of acetic acid bacteria, has received tremendous interest in various sectors including biomedical fields owing to its specific physico-mechanical properties. The unique properties of BC include a high water-holding capacity, slow water release rate, high crystallinity, biocompatibility, biodegradability, ultrafine web-shaped fiber network structure and the possibility to be molded into various forms during production. These intrinsic properties make BC excellent for industrial application especially in medical sciences.Moreover,the BC-nano-reinforced composite films can be made with superior activities. Therefore, we propose the current study to produce highly valuable biomedical material BC from wastes including sugar, juice, food and various other industrial wastes under static and shaking conditions by the use of micro-organisms.  The production of BC from chemically defined Hestrin–Schramm (HS) medium will be considered as control. The chemical structure of BC samples will be confirmed through Fourier Transform Infrared Spectroscopy (FTIR) and X-ray crystallography (XRD) diffraction analysis. Field Emission Scanning Electron Microscopy (FE-SEM) analysis will be performed to know about the fibril arrangement. Water holding capacity, mechanical and thermal properties will also be studied. The successful completion of this study will provide a base for the use of clean technology in Pakistan. Overall, this study will use clean technology to convert waste into highly valuable medical product which will improve wastes management and health condition of local community through reducing pollution and providing biomedical material. It will also better the economic condition by providing cheap source biomedical materials which will reduce financial burden on the health care system of the country.

Currently there are several kiwifruit cultivars belonging to Actinidiadeliciosa and Actinidia chinensis that have great genetic variation and are available for research and cultivation purposes.  This natural variation may be reflected in their differences in growth and adaptation to different environmental conditions.  In addition to other characteristic the genotypes belonging to these two groups are mostly separated vertically along the altitude with A. deliciosa usually adapted to higher and colder altitude at 800 to 1400 m and sometimes up to 1950 meters above sea level (MASL).  Contrarily,   A. chinensis mostly occurs at lower altitude between 200 to 900 MASL,   but can also be found at altitude as high as 1200 MASL.  A.deliciosa generally grows in area with low annual rainfall (600-1600 mm) and low relative humidity (60 to 80%) as compared toA. chinensis species that mostly grow in areas with higher annual rainfall (1000 to 2000 mm) and high relative humidity (75 to 85%).

Plant ecologists agree that the adaptation and distribution of plant species is strongly influenced by climate and in particular temperature and moisture regime. Variation in altitude often causes of changes in climatic factors including temperature, pre­cipitation, light intensity and nature of soil, which make the growing environment of plants more complex. Therefore, taking advantage of the altitudinal variations in the area of lower and upper extremes of Hazara division will provide good opportunities to evaluate the available kiwifruit germplasm/ varieties at NTHRI, for adaptation to different environmental condition. The altitude gradients in these areas vary from 832 MASL in Havelian tehsil of Abbottabad to about 2500 MASL in District Batagram of Hazara Division.  

Agriculture is the main source of income for most of the rural population of Hazara division.  Due to its agro-climatic conditions, Hazara Division has great potential for growing a variety of fruit crops such as apple, peach, pear, apricot, plum, cherries, walnuts and almonds which are high value cash crops. Thus cultivation of fruit occupies an important position in the farming system of the hilly area by enhancing household nutrition and is a major feature of subsistence economy. This farming system of the hilly area of Hazara Division is also most suitable for growing kiwi fruit. As kiwi is a species sensitive to waterlogging, it grows well on sloppy, hilly area where the soil is aerated and has good drainage system.

 NTHRI has been carrying out research and development activities on kiwifruit for the last three years in District Mansehra and has introduced the new fruit on farmer’s field. It is obvious from our last three years’ experience during which we observed that kiwi has vigorous growth and higher survival rate at NTHRI, Shinkiari Mansehra as compared to other low land area of Pakistan. However, we have so far tested only Hayward variety on farmer field in district Mansehra while the other varieties/germplasm also need to be evaluated not only in District Mansehra but also in other districts of Hazara Division before starting their cultivation on commercial basis. The new genotypes/germplasm have been acclimatized and their population has been increased at NTHRI nursery.  Theyhave greater genetic diversity with respect to plant and fruit phenotypes and adaptability to fluctuating environment.Thus NTHRI has great potential for propagating kiwifruit germplasm. However, to evaluate and promote kiwi plant germplasm/varieties in Hazara Division intervention is needed in this sector. The scope of kiwifruit cultivation, the availability of kiwifruit germplasm at NTHRI and the suitability of the agro-environmental condition of district Mansehra justify the evaluation of these germplasm along the lower and higher altitude of Hazara division before, their introduction/promotion on farmers field in the  areas.   

Currently, there is a continuous and growing concern about the food we daily eat, mainly the vegetables. Vegetables are packed of phytochemicals, minerals and vitamins due to which their role for health is significant.Antioxidant compounds such as polyphenolics play an essential part to scavenge the free radicals and inhibit the oxidative mechanism, which is responsible for degenerative health disorders. Allium cepa L. (onion/Piyaz) is an important vegetable crop, which is cultivated through the world mainly in Asia. It is one of the best nutritive sources of biologically active compounds. Many epidemiological studies have revealed that ingestion of A. cepa decreases the risk of cancer, cardiovascular diseases and neurodegenerative disorders. Furthermore, A. cepa possess antioxidant,anti-inflammatory, antimicrobial properties, which are mainly accredited to abundant contents of phytochemicals such as flavonoids, phenolic acids, organosulphur compounds, thiosulfinates, anthocyanins, vitamins and minerals (Fe, Se, K, I, S etc.). It is well established that climate, growing conditions, soil composition, post-harvest condition and genetic diversity effect significantly on the production of secondary metabolites or phytochemicals and properties in plant species related to active constituents. In Pakistan, A. cepa is cultivated under diverse agro-ecological conditions. However, impact of environmental conditions on secondary metabolites production and their properties has seldom been described. In this perspective, current study is designed to investigate the impact of environmental factors and genotypic difference on the phytochemical composition, in vitro antioxidant capacity and antimicrobial properties of A. cepa cultivated under diverse agro-climatic conditions in Pakistan. Proposed study will be helpful in understanding, impact of environmental conditions i.e. temperature, precipitation, altitude and genetic diversity on secondary metabolites or phytochemicals production and their activity in plant species, particularly in A. cepa. Furthermore, proposed study will be valuable for consumers, environmentalists, farmers and plant breeders to select the super varieties of A. cepa having maximum yield and significant contribution in health along with best growing conditions.

Description 

Aesthetic appearance has always been a matter of prime importance. Traditional medicinal resources, especially plants contribute significantly in the management of skin conditions. Herbal cosmetics, particularly obtained from plants are used for cleansing, beautifying, promoting attractiveness, coloring, softening or alteringthe appearance of body. Herbal extracts have been used to treat various ailments of skin, hair, and dental care and are primarily added to the cosmetic formulations due to antioxidant, anti-inflammatory, antiseptic, antimicrobial and anti-aging properties. Herbal or phyto-cosmetics claimed to have efficacy and intrinsic acceptability due to less side effects. The Himalayan region in Pakistan is biodiversity hot spot and flora of this area is being utilized by local communities as food and for the treatment of various health disorders including skin infections. The tribal women in Himalayas use local herbs as natural cosmetic and for the protection and caring of skin. And demand of herbal cosmetics is increasing due to their purity, little or no side effects and impressive results.

This project is mainly focused on the composition and properties analysis of medicinal plants of the Himalayan region of Pakistan. Though, several plant species are being utilized for skin care and in the treatment of many skin infections but have rarely been explored yet. Therefore, in this project: Phytochemical profiling of medicinal plants of Himalayan region of Pakistan will be done with special emphasis on the estimation of polyphenolic, flavone compounds and vitamins along with in vitro antioxidant/free radical scavenging potential and antimicrobial activity, particularly against pathogens causing skin infection. This project will be helpful in the screening of novel herbal/plant resources with significant potential to care and protect the skin. Further, findings of this project will be a milestone in the selection of plant species for the production of novel natural cosmetic products on commercial basis. 

Despite agricultural societies' primary reliance on crop plants, the tradition of eating wild plants has not completely disappeared. In developing countries millions of people do not have enough food and approximately, over 300 million people gain part or all of their livelihood and food from forests. Previous epidemiologic studies have consistently shown that, intake of fruits and vegetablesplay a crucial role in the prevention of chronic diseases including cardiovascular disease, cancer, Alzheimer disease, and age-related functional decline. This is attributed to the fact that these foods may provide an optimal mix of phytochemicals such as natural antioxidants and fibers along with other biotic compounds. Wild fruits can easily be managed, used and sold as they require very little input and management, but have been neglected by the scientific and development systems. Wild fruits are important source of nutrients, phytochemicals, vitamins and minerals to the rural populace; hence knowledge about their phytochemical contents and antioxidant potential is imperative. Many of wild edible fruits are scarcely known systematically, nutritionally, much less their biodiversity, phytochemical constituents or antioxidant potential throughout the world generally and in Pakistan particularly. Although Pakistan is an agricultural country but unfortunately we are not able to fulfill the food requirements of our people, in this regard wild fruits are the Nature’s gift for the inhabitants of rural areas. The Himalayan region in Pakistan is biodiversity hot spot area, particularly with reference to medicinal and edible wild food plant species. Proposed study will provide a pedestal (i) to scrutinize and identify edible wild fruits affluent in phytochemical constituents and antioxidant potential, (ii) in exploration of new and alternative food resources to improve food security of the nation, and (iii) for nutraceutical, beverages and confectionary industries of the country.    

Genetic Engineering of Lycopersicum esculentum for Enhanced Salt Tolerance through Cell Signaling Molecule involved in Multiple Aspects of Salt Tolerance

At present, 20% of global arable land is affected by salinity, and is estimated to exceed 50% by year 2050. Salt stress causes osmotic stress, ion toxicity and oxidative stress in plants. Being sessile in nature, plants have adopted various mechanisms to cope with salt stress. These include, but not limited to, synthesis of compatible solutes, ion compartmentalization, and detoxification of reactive oxygen species (ROS). Some plants have naturally adopted to withstand salt stress. On the other hand, most of the important crops do not posses significant degree of salt stress tolerance. Therefore, it is need of hour to develop salt stress tolerant crops through transgenic approaches. In this regard, plants possessing natural salt stress tolerance are great resource. Genes conferring salt stress tolerance could be identified and isolated from these plants and transferred to target crops to confer salt tolerance. Eruca sativa (Taramira) is known salt tolerant plant and is grown in drought and salt affected areas of Pakistan. However, biochemical and genetical basis of salt tolerance mechanism remained unexplored. Current project is designed to evaluate extent of salt resistance in Pakistani cultivars of Eruca sativa and to carry out detailed biochemical characterization of salt tolerance in most tolerant cultivar(s). To gain insight into genetical mechanism of tolerance, expression of a set of key genes known to be involved in salt stress response/ resistance will be determined. Seeds of E. sativa cultivars grown in different areas of Pakistan, will be acquired and plants will be initially screened for extent of salt stress tolerance on the basis of morpho-physiological parameters. The salt tolerance mechanism of most tolerant cultivar(s) will be comprehensively studied through biochemical assays. Expression of key genes involved in osmoprotectants synthesis, stress-induced hormones, ion channels/ transporters, and stress induced transcription factors will be determined to gain insights into differential molecular responses.  The proposed research will be helpful in understanding biochemical and molecular strategies of salt tolerance adopted by E. sativa. The clear understanding of molecular strategies adopted by naturally salt tolerant plants could be helpful in developing salt stress tolerant crops in future.

Phosphoenol pyruvate carboxylase (PEPC) is a ubiquitous cytoplasmic enzyme present in vascular plants and is also widely distributed among archeal, bacterial, cyanobacterial and unicellular green algal species. PEPC is clearly best known for its cardinal roles in C4- photosynthesis and CAM. Most of the vegetative, fruit and crop plants are C3 plants. The main problem with C3 plants is that they can’t tolerate the harsh conditions like drought and hot climates. C4 and CAM plants have special adaptations that enable them to survive in the hot and dry conditions. C4 and CAM plants have mechanism to control the photorespiration. One of such adaptation is the presence of enzyme PEPC. This enzyme catalyzes the irreversible β-carboxylation of PEP in the presence of HCO3 and Mg2 to yield oxaloacetate (4-C compound) and inorganic phosphate. Then 4-C compounds are transported to bundle sheath cells where bound carbon is released again as CO₂. One of the challenges that plant biotechnologists are facing is to modify the photosynthesis of C3 plants in order to achieve the increase in net carbon gain. Initially they were interested in engineering RuBisCo in order to increase its carboxylase activity as compare to oxygenase activity, but it has not been possible yet. Today main interest is to transfer the C4 traits in C3 plants to improve their photosynthetic characteristics and to make them stress tolerant. In modern day genetic engineering the Agrobacterium tumefaciens mediated gene transfer system is widely used to transform the plants. In current project, our aim is to transform local variety of tomato by cyanobacterial pepc gene using the Agrobacterium tumefaciens mediated gene transfer system. In the long run we intend to develop tomato plants with C4 plants like characteristics that demonstrate better growth and development in harsh environments.

Aflatoxins are those organic compounds which are produced by the fungus on feed materials. Aflatoxin B1 is one the most hazardous aflatoxins and humans as well as poultry both are severely harmed by this toxin. It is necessary to degrade/eliminate mycotoxins from the poultry feed to ensure poultry health. The current study is designed to isolate the bacteria from the various sources which will have capability to degrade the aflatoxin B1. Isolated bacterial strain would be identified biochemically. Shake flask experiments would be carried out to determine the rate of degradation. TLC and HPLC would be the analytical method to study degradation. Co-metabolism degradation of afltatoxin B1 by newly isolated strain would be studied by supplying different sugars as extra carbon source.

Wheat is staple food and important source of energy and nutrition for mankind. However wheat is suffering major yield losses due to various biotic and abiotic stresses. Worldwide food problem drew the attention of scientists to develop new methods for improving genetic makeup of plants. Various methods are available to produce transgenic plants; plants with desired characters and of approved quality. Amongst these, two methods are most widely used; the Agrobacterium-mediated and microparticle bombardment methods. The Agrobacterium-mediated transformation method has several advantages over the microparticle bombardment method. The former method results in more stable integration of a defined segment of DNA into the plant genome, lower copy number and fewer rearrangements in plant genome. Although Agrobacterium-mediated cereal transformation via tissue culture brought some success but it involves several limitations; it requires sterile conditions and is time-consuming. Moreover, the tissue culture approach causes somaclonal variation due to either epigenetic effects or chromosomal rearrangements. Many cereal crops including wheat have been successfully transformed through Agrobacterium-mediated method. But still transformation of wheat lag far behind than rice and maize due to problems of regeneration of some varieties .To overcome this sort of problems in planta transformation method has been introduced, through which intact plants are transformed and there is no need of in vitro culturing. In this method soaked seeds of plants are pricked with a needle inoculated in Agrobacterium solution. The inoculated seeds are allowed to mature into seedlings and transferred to plant pots and exposed to natural environment rather than controlled conditions and allowed to pollinate freely. As wheat is an important crop, our aim is to transform local wheat varieties with this simple in planta transformation method. This approach will provide an alternative way for genetic improvement of the varieties having low efficiency of regeneration and hence difficult to transform through tissue culture technique.

The Tagetes minuta is wildly grown in abundant in Pakistan especially in hilly areas. Hazara division is a rich source of this important species.  The plant species is assumed to be containing very important compounds of medicinal nature.  The aim of current project will be to isolate and analyse some of the important compounds from Tagetes found in Hazara Division.  Qualitative analysis will be performed using thin layer chromatography(TLC) techniques. Further purification will be done using column chromatography followed by determination of biological activities of the purified compounds. Particularly we will be interested in antibacterial, insecticidal and phytotoxicity related biological activities of the purified compounds from Tagetes. These results will provide basic information about the compounds present in this important and wildly grown plant species that will further our knowledge to identify the structure of the compounds which will eventually be useful in drug discovery.

Naturally occurring plants possess great biological and medicinal importance that is effective against various            diseases. Arisaema is very important, biologically active medicinal plant. The plant species is assumed to be containing significant biochemical compounds of medicinal nature. It is therefore necessary to evaluate some of the important compounds from this plant species as it has not yet been fully explored for its chemical constituents. The purpose of current study will be to isolate and characterize the flavonoids especially flavonols and anthocyanins fromArisaema found in North and Northwestern parts of Pakistan. Qualitative analysis will be performed by using the thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) techniques. Further purification and isolation will be done by using column chromatography followed by determination of antimicrobial activity of plant crude extracts and purified compounds from Arisaema. The results will provide the basic information about flavonoids present in this important plant species that will further our knowledge to identify the structure of flavonoids which will eventually be useful in drug discovery.

Both natural environmental processes and anthropogenic activities are responsible for increased rate of contamination in biosphere. Deleterious heavy metals are bioactive and potentially toxic to plants, animals and humans. These pollutants affect organisms on every biological level and leading to toxicity from molecular to ecosystem scale. Heavy metals induced oxidative stress as a result reactive oxygen species (ROS) are generated which are widely implicated in cell membrane and DNA damage.  Arsenic (As) and Lead (Pb) are bioactive toxic metals present in the environment in both organic and inorganic form. Pb and As contamination due to industrial activities such as mining and smelting, energy and fuel production, fertilizers and pesticide application, and exhaust emission from vehicles is increasing day by day.Pb contamination of water and soil is potentially toxic for both plants and human health due to persistence of Pb for long time in air due to soil erosion. Interactions of these metals interrupt several physiological and biochemical processes in plants and could induce genotoxicity events. The plants grown in contaminated soils have elevated level of heavy metals and being non degradable. They enters in food chain via different routes can be passed to various organisms causing many lethal diseases.Developments of PCR based techniques are most important for analysis of the mechanisms of heavy metal tolerance in plants at a biochemical and molecular level. Currently DNA marker techniques are applied to infer all routes through which toxicants may affect the genetic structure of exposed plants/organisms. The random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) assays are PCR-based techniques and competent of detecting point mutations, temporary alteration of DNA and detection of low doses of pollutants. The changes in RAPD/AFLP profile can be evaluated as alteration in genomic stability test (GTS). The present study is designed to see heavy metal induced changes in Tagetes using RAPD/AFLP assay. Changes in RAPD and AFLP profiles will be compared to biochemical parameters (chlorophyll, carotenoids, proline and Malondialdehyde (MDA) content). Results obtained may suggest that molecular, physiological and biochemical assays could be used together as reliable and powerful biomarkers to determine genotoxic effects of heavy metals in ecotoxicology.

Most of the world’s population relies on medicinal plants for their primary health care. Plant based medicines have reached widespread acceptability as therapeutic, nutraceutics, and cosmetic agents. Medicinal plants contain variety of bioactive compounds particularly phenolic compounds that are reported to have potential antioxidant properties. Flavonoidsas natural antioxidants are abundant in plants and these play vital role in health care.It is generally observed that humans can accept naturally occurring materials more easily in comparison to synthetic compounds. There is thus a strong need for exploring effective antioxidants from natural sources to prevent deterioration of foods. Pakistan has great potential for drug discovery through the use of natural products abundantly present in medicinal plant flora.  Due to its geographical and climatic conditions Pakistan captures a unique position of having a wide diversity of medicinal plants growing over a range from cool temperate to humid and hot tropics.  These varied edaphic and climatic factors of the country reflect diversity and valuable medicinal heritage. Five medicinal plants species including four species of Roscoea i.e., R. capitata, R. auriculata,R. alpine,R. purpurea and one species of Dicliptera D. bupleuroides would be selected from Northern regions (Hazara Div, Margalla and lesser Hamalaya) for isolation and characterization  of flavonoids (flavonols, anthocyanidins, flavones, flavanones, catechins and isoflavones). These plants hold significant medicinal importance and are rich in bioactive components. The proposed project will analyze total flavonoids in plant extracts. The antioxidant activity of extracts and isolates will be evaluated by radical scavenging assays. By the completion of the proposed project, these natural resources will be conserved with derived products useful for health.  Additional benefits would be to support pharma, food and biotech industries through potential products and processes. 

Horticultural crops are highly perishable in nature and quickly deteriorate after harvest due to inadequate postharvest handling practices and non-availability of storage facilities. In developing countries like Pakistan, postharvest losses are particularly high in case of summer fruits and vegetables, since temperature is the major determinant in the shelf-life of fresh produce. Refrigerated storage is the best option but is energy extensive, requires huge initial investment and is not easy to install. Moreover, existing energy crises in Pakistan limits the adoption of cold storage technology. Small land holdings and financial constraints of growers confine the use of high capital venture. In the absence of proper storage facility, the farmers usually sell their produce in local market soon after harvest.Low cost ecofriendly Zero Energy Cool Chambers (ZECC) are currently being used in many countries including India, Japan, Tanzania and Ghana for on-farm storage of horticultural produce. However, this technology has not been introduced in Pakistan yet. Vehari is located in Southern Punjab where socioeconomic condition of farmer is poor and most of the farmers are vegetable growers. Moreover climate of Vehari is also hot and dry which results in more postharvest losses to the small scale growers who did not afford expensive cold storage facilities. To address these issues a low cost, ecofriendly ZECC will be constructed at COMSATS Institute of Information Technology Vehari, Campus.In ZECC, moist sand is filled between two brick walls, one nested inside of the other. Warm air, picks up the water molecules from moist sand and increases humidity in the storage chamber.Horticultural crops (fruit, vegetables and flowers) along with various postharvest treatments (packaging materials and chemical treatments), will be kept in ZECC as well as in ambient conditions in laboratory. Shelf-life performance and physico-chemical quality will be evaluated and the data will be statistically analyzed. Costs benefit ratios based upon the cost of ZECC construction (one time for 2 years) using local materials and benefits such as reduced losses and/or increased market value will also be determined. Outcome will also be communicated through a concluding seminar at the end of the project to disseminate this technology to farmers and retailers. In future ZECC will be used for demonstration and for executing various research trials.