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National Institute for Genomics and Advanced Biotechnology

ACHIEVEMENTS

Transgenic Plants (GMOs) Developed

  • Rust resistant transgenic wheat line

  • Drought and salt tolerant wheat lines

  • Drought and salt tolerant rice lines

  • Herbicide resistant Groundnut transgenic lines

GMO testing

  • First survey confirming the presence of Bt cotton in field growing areas of Pakistan based on laboratory detection in 2007-2009

  • Establishment of Bt gene authentication and expression monitoring system in cotton

  • Genetic transformation protocols mediated by Agrobacterium tumefaciens have been successfully established in local rice, wheat, groundnut and potato

  • Yellow rust is one of the major causes for decline in the production of wheat in Pakistan. Agrobacterium tumefaciens mediated transformation protocol has been established by using mature embryos as explant source in local varieties, which include Inqilab-91 and Chakwal-97. Using the optimized protocol, rust resistant gene has been successfully incorporated into Chakwal-97. Bio-assays have demonstrated the resistance of transgenic plants against pathogen.

  • Drought and salt tolerant genes successfully transformed in wheat varieties Lasani and Tatara.

  • DREBIA regulatory element for drought and salt tolerance under the control of 35s and lip promoters has been successfully transformed into rice variety JP-5. Transgenic rice plants are under evaluation process .

  • A survey was conducted in the cotton growing areas of Sindh and Punjab provinces to assess the status of Bt cotton during July-August, 2007-08 and June-July 2009-10. 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. The survey also revealed the presence of Bollgard-I event only in Pakistan

  • 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.

  • 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.

  • Protocol for callus induction, regeneration system and Agrobacterium mediated transformation has been developed, Chitinase gene for rust resistance has been successfully transformed in wheat verities. The molecular analysis (PCR) confirmed the presence of chitinase gene in transgenic wheat plants.

  • Major vernalization and photoperiod response genes of 60 Pakistani wheat varieties have been identified. Different combinations of the major vernalization genes were found in these varieties. All varieties except “Era” had the Photoperiod insensitive allele at Ppd-D1a. The difference in flowering time of the studied varieties was not explained by the combinations of different alleles at the major vernalization loci. Earliness per se seems to be the major determinant of flowering time in Pakistani wheat varieties.

  • Sex in Papaya cannot be determined morphologically before flowering. We screened 40 Papaya plants of Pakistani varieties with 5 pairs of SCAR markers including T12, W11, SDP, PKBT-4 and PKBT-5. Primer pairs SDP, W-11 and T12 clearly differentiated between male and female plants of the studied papaya plants and can, therefore, be used for sex determination at seedling stages.

  • Molecular characterization of Pakistani wheat with gene specific markers showed that very few Pakistani wheat varieties namely, Lyallpur-73, Khyber-87, Bakhtawar-92, Suleman-96, Saleem-2000, Wafaq-2001 and GA-2002 carry Lr34/Yr18. Of the 24 advanced wheat breeding lines of Wheat Program, NARC, 10 had Lr34/Yr18.

  • Salinity and drought stress-responsive genes are being identified in rice and wheat in comparison with a halophyte Salicornia.

Future Thrust

  • Genetic transformation of cotton against sucking insect pests and CLCV.

  • Genes Identification for biotic and abiotic stresses.

  • Identification of genes through proteomics approaches.

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

  • Genetic transformation of chickpea for biotic stress tolerance.

  • Identification and isolation of new plant gene promoters.

  • Isolation of genes against sucking insect pests.

  • Identification and functional characterization of salinity and drought-responsive genes in rice and wheat in comparison with Salicornia

  • 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.

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

  • Identification and molecular characterization of economically important microbes.

  • Disease free pre-basic III seed of potato will be made available to the potato growers in Pakistan.

  • Hybrid seed production cycle will be shortened

  • Female Papaya in-vitro plants will be made available for mass scale plantation.

  • Molecular characterization of Pakistani wheat varieties for important agronomic and quality traits.

  • Improvement of crop plant productivity through radical crop design

  • Functional genomics by using high throughput techniques (ChIP-SEQ, ChIP-CHIP tilling array, Yeast 2 hybrid) for the functional characterization of genes and their regulation involved in floral organ identity in model and crop plants.

  • Functional evolution of genes involved in floral organ identity

  • 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 using SSR markers.

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

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 for drought, salt tolerance disease resistance have been successfully transformed into rice, wheat and groundnut via Agrobacterium mediated method. Protocols For the optimization of protocols for cotton chickpea, lentil and tomato are in progress.

GUS expression analysis in rice leaf tissue   DREB1A Gene for drought/salt tolerance

 

 

 

     

Disease resistant transgenic wheat  

Non transgenic wheat

 

Development of protocol for transgenic groundnut

GMO Testing

An effective has been established for Bt gene authentication, quantification of Bt toxin level, marker gene detection has been established.
 

Functional evolution and genomics

The focus of our research is study functional evolution and genomics in plants. Elucidating molecular mechanisms underlying evolution of morphological novelties and functional genomics of gene involved is the major objective. In collaboration with research group at Max Planck Institute for Plant Breeding Research, Cologne Germany and state Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, China, we have characterized genes responsible for calyx inflation and floral transition in Solanaceos plants

 

Tissue culture

Tissue culture team is mainly involved in production of virus-free potato and banana plants through tissue culture and micro propagation.

 

POTATO

Virus free seed is produced through tissue culture technology and disseminate with the co-operation of potato seed growers and National Potato Programme. Resistance against fungal diseases will be induced through genetic transformation. For this purpose protocols for transformation of chitinase gene have already been optimized for variety cardinal and will be exploited for other commercial varieties to produce transgenic potato. 
 

ACHIEVEMENTS

Virus free nucleus seeds of potato are being produced at NARC green houses. During the year 2009 and 2010 Seventeen thousand nucleus seed has been harvested from green house at NARC. These nucleus seed will be further multiplied at northern areas. In the year 2009-10 the field trails were conducted at Summer Wheat Research Station Kaghan and Sindh (Mir Pur Khas) and results were satisfactory.



Potato Seed Production at NARC

 


Potato Plantation at Mir Pur Khas Sindh

 

Hybrid Plant Production through Embryo Rescue

In vitro embryo rescue techniques are being often used to rescue plant embryos from aborting progeny seeds that result when two distantly related plants (e.g. two species) are crossed together. Such ‘wide crosses' are often desirable to transfer genetic traits from wild relatives to cultivated crop plants. The embryo rescue technology is always helpful in plant breeding or hybrid seed production programs.

 

Objectives

• Obtain the three to four generation in one year to reduce the generation gap.

 

Achievements

  • Initially different explants (mature and immature seeds) of nonspecific crosses have been collected from respective crop Programs for optimization of tissue culture protocols. Different tissue culture media (MS, B5 and N-6) with different growth regulators (Benzyleaminopurine, Indoleacetic acid and Gibberrellin @ 01-2.0 mg/l) were used. The explants (embryos) were excised after different time span ranging from 15 days to one month of after pollination.

  • Seeds of wheat and maize were soaked in distill water for 24 hrs before embryo excision. The embryos of wheat variety NARC-2009 has shown the high regeneration rate on MS medium under dark while the embryos of maize has shown high regeneration on B-5 medium.

  • Seeds of nonspecific crosses of canola 30 DAP has resulted higher regeneration as compare to 15, 20 and 25 days old DAP. Embryo excised from maize genotype, Karamet, R2208, R2303, Pan 610, Pan 590 and PSCH 02 started germination after 4th day of culturing on B-5 medium containing MS vitamins.

Wheat plantlet formation from embryo culture

 

Maize plantlet formation from embryo culture


Brassica premature embryo culture


 


Tomatoes premature embryo culture

 

 

Micropropgation of Papaya through Tissue Culture

Papaya is dioecious, hetrozygous and cross pollinated specie. Conventional propagation of papaya through seed resulted high rate of variation in fruit quality. There is genuine need to provide tap alternate nonconventional method for propagation like tissue culture. There are several reports of papaya multiplication through tissue culture by using shoot tip and axillary bud as an explant from the mother plant. But there are only few reports on the micropropagation of papaya.

 

Progress

Micro-propagation of female papaya (cv. Cinta) was carried out using axillary bud and shoot tip. Explants were excised from six month old field grown plants. Female plants were selected after bearing flowering and fruiting. Explants were culture on MS media containing 0.1 mg/l IAA and different concentrations of BAP (0.0-2.0 mg/l).

 

In vitro multiplication of papaya

 

 

Banana production through tissue culture

In vitro Multiplication: In vitro multiplication was started in November 2009. Initially four cultivars (dwarf cavandish, pisang, Williams hybrid and Brazillian) were focused for plant production. Total number of in vitro plants produced up to 31st March,2010 is more than 10,000.
 

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.
 

 

Isolation 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 will be 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.

 
 

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