PLANT BIOTECHNOLOGY PROGRAMME
NARC, Islamabad

INTRODUCTION
OBJECTIVES
RESEARCH ACTIVITIES (On-Going)
PROJECTS
ACHIEVEMENTS
FUTURE THRUST
HIGHLIGHTS OF RESEARCH

• Genetic Engineering of Crops

• Tissue Culture

• Developing Host Plant Resistance for Bacterial Blight (Xanthomonas oryzae) in Rice

• Development of New Plant Type in Rice by Double Haploid Breeding

• Marker-Assisted Selection and QTL Mapping

• Marker-assisted Selection for Biotic and Abiotic Stresses in Wheat

• Identification of Genes involved in Iron and Boron Nutrient transport in Plants

HUMAN RESOURCES DEVELOPMENT
RECENT PUBLICATIONS
RESEARCH TEAM

INTRODUCTION
Tissue Culture Laboratory was established at NARC in 1982 by Late Dr. Azra Qurashi. The laboratory is known to be the pioneer tissue culture facility in the country with emphasis on pre-basic virus-free potato seed and producing clones of other crops like, date palm, rice, carnation and banana. The laboratory earned a name in production of disease-free potato seed and banana plants. Later, the lab was upgraded and renamed as Plant Biotechnology Program (PBP) and it was merged with the Institute of Agri-Biotechnology and Genetic Resources (IABGR) during 2001. Plant Biotechnology Program has been strengthened by consolidation of the available expertise presently scattered at National Agricultural Research Centre and foreign qualified scientists in the subject have been pulled from various programs. Presently, the program is housed in the Plant Genetic Resources (PGRI) building.

A National Institute for Genomics and Advanced Biotechnology (NIGAB) has been established at NARC and it will have two separate components; Plant Biotechnology Program, and Animal Biotechnology Program. Introduction of modern research technologies and problem-oriented research objectives form basis of the program. Cutting edge technologies used in the area of DNA recombination, transcriptomics, proteomics marker-assisted breeding and genetic transformation is being employed for gene isolation, diagnostic tests and production of transgenic plants. Efforts are underway to upgrade the physical infrastructure and human resources development under the umbrella of NIGAB. Construction of new buildings for research labs and greenhouse experimentation facilities are in progress. Well qualified scientists have been engaged in planning and execution of research projects and building of infrastructure. Regular training and interaction of scientists with national as well as international research and academic establishments is integral part of the planning. Main thrust of the program is to develop environmentally safe local crop genotypes with economically important traits. Marker-assisted breeding and QTL mapping is another area of our research priority.

OBJECTIVES

• Molecular biological research on specific problems of economic importance in selected crop plants and microbes.

• Identification and characterization of important genes (biotic and abiotic for stresses tolerance) from crop plants and microbes and their exploitation through functional genomics.

• Screening and evaluation for salt tolerance using micro satellite markers and QTL mapping in selected crop plants.

• Establish species- and variety-specific tissue culture protocols for efficient regeneration of transformed calli and tissues.

• Search for new genetic resources carrying economically important traits.

• Data management and development of bio-informatics tools.

• Human resource development in plants and microbial genomics.

RESEARCH ACTIVITIES (On-Going)

• Developing high frequency regeneration system and transformation in rice and wheat.

• Genetic improvement of wheat and rice for drought and salt tolerance through marker-assisted selection and genetic transformation.

• DNA finger printing of traditional and improved cultivars of wheat and rice.

• Genetic transformation of potato for induction of disease resistance against fungal pathogens and production of disease free potato seed through tissue culture.

• Transgenic Tomato with resistance to bacterial wilt.

• Production of virus free banana plants through tissue culture. Status of cotton harboring Bt gene in Pakistan.

• Production of inter-specific crosses in rice through embryo rescue.

• Marker-aided selection of rice genotypes for resistance to bacterial blight in rice.

• Transgenic plant production of rice for bacterial blight resistance gene.

• Identification and molecular characterization of important microbes.

PROJECTS

ACHIEVEMENTS

• Banana crop in Pakistan is under severe threat of banana bunchy top virus (BBTV). The only way to replace infected fields is by healthy banana plants. More than one thousand disease free banana plants were produced through tissue culture technology and tested at different locations in Sindh. Banana plants have been transformed with marker gene while induction of resistance against BBTV is in progress.

• Production of pre-basic seed potato through tissue culture. This technology was disseminated to the provincial research institute, farmers and the private sector. The cumulative impact of the technology resulted in the decline of seed potato imports. Chitinase (antifungal) gene has been successfully transformed in potato variety cardinal and transgenic plants showed complete resistance to Fusarium and partial to Phytophthora. The molecular analysis revealed the presence of chitinase gene in transgenic potato plants.

• Agrobacterium mediated transformation has been achieved in commercial grown cultivars of tomato (Roma) for bacterial wilt resistance.

• Yellow Rust, drought and high temperature are major causes for decline in the production of wheat in Pakistan. Agrobacterium tumefaciens mediated transformation protocol has been established by using two days mature embryos as explant source in local varieties, which include Inqilab-91 and Chakwal-97.

• Bacterial leaf blight caused by Xanthomonas oryzae (Xoo) is one of the major bacterial destructive diseases of rice, which causes loss of 50%. This is soil born and chemical control is difficult. We transformed rice Basmati-385 with Xa21 gene. The transgenic lines have been produced which are under evaluation.

• A survey was conducted in the cotton growing areas of Sindh and Punjab provinces to assess the status of Bt cotton during July-August, 2007. Ten districts in Sindh and 11 districts in Punjab were surveyed and samples were collected from 126 various field sites. In laboratory, ImmunoStrip analysis and ELISA revealed that 80% samples from Sindh and 90% samples from Punjab were positive for the presence of Bt-Cry toxin.

• Protocol for inter-specific crosses was developed using embryo rescue technique and three wide crosses were produced across the incompatibility barriers: IR6 x O.nivara, Basmati 385 x O.nivara, KS282 x O.nivara.

• More than 150 genotypes of rice and wheat were tested under hydroponic condition using different salinity levels. The same material is being tested in the hot-spot areas of salinity. The best performing genotypes will be subjected to microsatellite markers with the hope to get linkage.

• Mapping populations are being developed for QTL analysis for salt, drought and rust resistance in wheat.

FUTURE THRUST

• Genetic improvement of wheat and rice for drought and salt tolerance through marker assisted selection and genetic transformation.

• QTL mapping for salt tolerance in wheat and rice using SSR markers.

• Identification of new SSR markers for placing them on the already developed genetic linkage maps.

• Identification of genes through proteomics approaches.

• Identification of genes involved in iron and boron transport using molecular techniques.

• Construction of cDNA library of wheat crop for traits of economic importance.

• Sequencing of the cDNA library and expression analysis of selected genes through DNA chip technology.

• Genome sequencing of important bacterial species.

• Incorporation of gene from wild rice into cultivated for resistance to bacterial blight through embryo rescue and marker aided selection.

HIGHLIGHTS OF RESEARCH
Genetic Engineering of Crops
Main emphasis is focused on development of economically important transgenic crop plants like wheat, rice, tomato, potato and chickpea. Genes conferring drought, salt, rust, bacterial blight and other disease resistance are being used to develop transgenic crops through Agrobacterium-mediated transformation method. Development of disease resistant rice, tomato and potato are typical examples.

Tissue culture
Tissue culture team is mainly involved in production of virus-free potato and banana plants through tissue culture and micro propagation. Virus resistant potato and BBTV resistant banana have been developed through micro propapagation and are ready for commercialization.

Developing host plant resistance for Bacterial blight (Xanthomonas oryzae) in rice
Bacterial blight is a major disease of rice in Pakistan. Host plant resistance is the most effective method of controlling this disease. Local virulent bacterial isolates identified. Using these isolates, three wild species were identified as highly resistant to all the prevailing isolates of bacterial blight. Wide hybrids among susceptible cultivated and resistant wild species across the incompatibility barriers have been produced. Backcrosses are in progress. Molecular markers will be used to identify new sources of resistance and select segregating population.

Development of New Plant Type in Rice by Double Haploid Breeding

Marker-Assisted Selection and QTL Mapping

• Using DNA fingerprinting approaches for estimation of genetic diversity among different genotypes/cultivars of wheat and rice using RAPD, AFLP and SSR markers.

• Marker-assisted selection (MAS) for biotic and abiotic stress tolerance in wheat and rice.

Marker-assisted Selection for Biotic and Abiotic Stresses in Wheat
Wheat is the most important world cereal. In the face of biotic and abiotic stresses, increasing its productivity to feed ever growing population is a challenge for Agricultural Research. New emerging sciences of genomics and biotechnology promises much in this regard.

In our laboratory, we are trying to develop a system of marker-assisted selection for improvement in salt and rust resistance. For this purpose, QTL mapping populations are being developed. The extreme parents i.e. tolerant and susceptible, are being screened out of a large collection of germplasm at the Plant Genetic Resources Research Program at NARC. The germplasm is being tested at germination, seedling (in hydroponics) and reproductive stages (at three of hot spots of salt affected soils).

The mapping populations for salt and drought tolerance will be genotyped with SSR markers and QTL mapping will be done. Closely linked markers with the genomic regions controlling these traits of interest will be used for marker assisted selection of superior genotypes against these stresses.

Identification of Genes involved in Iron and Boron Nutrient transport in Plants
Iron (Fe) and boron (B) deficiencies are very common micronutrient disorders in field and horticultural crop plants grown on calcareous soils of Pakistan. In fact, majority of Pakistani soils are calcareous in nature.

A widespread and severe iron deficiency has been reported in Pakistan in a number of field as well as horticultural plants. Soil and plant factors affect the availability, uptake and translocation of the nutrient. Iron is abundant in Pakistan soil, however, due to high soil pH, it is found in ferric form which is sparingly available to plants defective in ferric iron solubilization/chelate release and uptake system. Dicots and non-cereal monocots, except rice, take up iron in ferrous form (Fe+2). This involves enzymatic reduction of ferric iron (Fe+3) to ferrous iron (Fe+2) at root surface. Rice possesses both systems of iron uptake i.e. it releases ferric iron chelators (phytosiderophores) and takes Fe+2 iron from the environment. The recent advances in molecular plant nutrition have revealed that the mechanisms are genetically controlled. Therefore, genetic modification of the affected crop plants offers great promise for growing healthy plants. As a first step, genes encoding ferric iron reducing proteins are being isolated from moss (Physcomitrella patens; http://moss.nibb.ac.jp/) and transferred into a groundnut plant.

Similarly, B deficiency is another nutrient deficiency problem limiting crop yields in the country. Plants defective in B uptake and/or translocation suffer severely by B deficiency. Improving B uptake and internal utilization by genetic manipulation afford several advantages over other methods of B deficiency correction. Due to narrow range of plant B requirement, not only B deficiency but also its toxicity is of considerable importance. Plants grown in extreme environments like saline and alkaline soils and fields irrigated with B loaded saline waters may suffer from B toxicity. Boron toxicity is still a neglected issue in Pakistan. Recently, microorganisms have been isolated from soils which are highly B and NaCl salinity tolerant. An attempt is being made to isolate these genes and transfer these characters to the plants grown in saline and alkaline soils. A strategy is also on cards to localize these genes on strategic locations in plant to coup with B deficiency.

HUMAN RESOURCES DEVELOPMENT
Biotechnology program is also focusing on human resources development, trainings, workshops, courses; number of students is being trained in the field of agricultural biotechnology.

A. Partial list of internees with duration of internship, etc (2007-08):

B. Partial list of Names and details of students who worked for M.Sc./Ph.D research (2007-08):

RECENT PUBLICATIONS