pakistan journal of botany

ISSN 0556-3321 CODEN PJB 0B6

PAKISTAN JOURNAL OF BOTANY A BIMONTHLY PUBLICATION OF PAKISTAN BOTANICAL SOCIETY

Volume 38 No. 5 (Special Issue), December, 2006 Guest Editors Muhammad Ashraf Department of Botany, University of Agriculture, Faisalabad, Pakistan And

Habib-ur-Rehman Athar Institute of Pure and Applied Biology, Bahauddin Zakariya University, Multan, Pakistan Chief Editor: Dr. Abdul Ghaffar Department of Botany, University of Karachi, Karachi-75270, Pakistan. E-mail. [email protected] Subject Editors: Plant Biosystematics, Taxonomy and Evolution -Dr. M. Qaiser Department of Botany, University of Karachi, Karachi-75270, Pakistan. E-mail: [email protected] Plant Biochemistry and Physiology -Dr. M. Ashraf Department of Botany, University of Agriculture, Faisalabad, Pakistan. E-mail: [email protected] Ecology, Phycology and Biodiversity -Dr. Farrukh Hussain Department of Botany, University of Peshawar, Peshawar, Pakistan. E-mail: [email protected] Cell Biology, Genetics and Microbiology -Dr. Shahida Hasnain Department of Botany, University of Punjab, Lahore, Pakistan. E-mail: [email protected] Mycology and Plant Pathology -Dr. Abdul Ghaffar Department of Botany, University of Karachi, Karachi-75270, Pakistan. E-mail: [email protected] Editorial Committee: Dr. S.I. Ali, Dr. Mustafa Shameel, Dr. S. Shahid Shaukat (University of Karachi), Dr. Iftikhar Ahmed (PARC, Islamabad), Dr. Kausar A. Malik (PAEC, Islamabad), Dr. Javed Iqbal (Punjab University, Lahore), Dr. Riaz H. Qureshi (University of Agric., Faisalabad), Mr. I.C. Hedge (Royal Botanical Garden, Edinburgh, UK), Dr. Klaus Lüning (Stiftung Alfred-Werger-Institut, Germany), Dr. Irwin A. Ungar (Ohio University, USA), Dr. M.A. El-Wakil (University of Mansoura, Egypt)

Pak. J. Bot., 38(5): 1599-1604, 2006.

DNA-BASED GENOTYPING OF SORGHUM HYBRIDS NABILA TABBASAM, MEHBOOB-UR-RAHMAN* AND YUSUF ZAFAR Plant Genomics & Molecular Breeding (PGMB) Lab, National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Jhang Road, Faisalabad, Pakistan Abstract Over the last two decades, DNA fingerprinting have been increasingly used to characterize germplasm of different organisms including crop plants, which was otherwise difficult with morphological and or isozyme markers because of paucity of genetic information at phenotypic level. In the present study, two hybrid samples of sorghum (Sorghum bicolor L.) received from Federal Seed Certification and Registration Department (FSC&RD), Islamabad Pakistan for DNAbased typing to prove their identity. A DNA fingerprinting technique, random amplified polymorphic DNA (RAPD) analysis was applied to find DNA polymorphisms using 20 different 10-mer primers. Out of these, 8 (40%) primers detected polymorphism between the hybrids. However, rest of the primers produced monomorphic pattern. Out of the 121 amplified loci, 33% were polymorphic. These polymorphic primers distinguished the sorghum hybrids. Our results revealed that RAPD is a useful tool in the identification of hybrids. In future, it can be utilized to estimate the extent of genetic diversity and warranting genome mapping studies in sorghum. Keywords: Sorghum, bicolor L., hybrids, genotyping, RAPD

Introduction Sorghum (Sorghum bicolor (L.) is ranked fifth after the four major grain crops (wheat, maize, rice, and barley) in the world, and is the second most important cereal crop (after maize) in sub-Saharan Africa. It was originated from Northeast Africa (the Ethiopia, Eritrea area), the center of diversity for the crop (Agrama & Tuinstra, 2004). Sorghum is cultivated mostly in the developing world (especially in Africa and Asia), although it has become an important industrial crop in many developed countries. Its value in arid climates is due to its ability to withstand dry conditions. In Pakistan, major usage of sorghum crop accounts for feeding livestock. The continued release of new crop cultivars, particularly F1 hybrids, by public and private breeders there is a need to test purity or identify duplicated crop varieties including hybrids. Traditionally, genetic purity determinations have been carried out by the evaluation of morphological or physiological traits expressed by seed, seedlings or mature plants. Seed observations and field-testing procedures are often inaccurate because environmental stress conditions during seed or seedling/plant development can mask the expression of specific morphological or physiological traits McDonald (1995). DNA markers are being increasingly utilized for the identification of hybrid, breeding lines and clones (Paul et al., 1997; Sonnate et al., 1997; Barrett & Kidwell, 1998; De-Bustos et al., 1999; Asif et al., 2005). DNA markers are considered constant landmarks in the genome. Markers are identifiable DNA sequences found at specific locations of the genome and transmitted by the standard laws of inheritance from one generation to the next. There are many different kinds of molecular markers including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), *

Corresponding Author: Mehboob-ur-Rahman [email protected]; Fax: +92-41-2651472

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amplified fragment length polymorphisms (AFLPs), microsatellites, and single nucleotide polymorphisms (SNPs). There are merits and demerits of each type of marker, depending on the specific objectives of the particular study. Among these, RAPD analysis is quick (Colombo et al., 1998; Mukhtar et al., 2002) and well adapted for nonradioactive DNA fingerprinting of genotypes (Karp et al., 1997; Cao et al., 1999). RAPD markers (Williams et al., 1990; Welsh & McClelland, 1990) have been widely used for genotype identification in plants (Rahman & Zafar, 2001; Mukhtar et al., 2002), to study genetic variation in various organisms, including crop plants such as tomato (Klein-Lankhorst et al., 1991), wheat (Devos et al., 1992), potato (Quiros et al., 1993) and sorghum (Tao et al., 1993; de Oliveira et al., 1996; Menkir et al., 1997; Ayana et al., 2000), for genetic purity determination in seed testing (Hashizume et al., 1993; McDonald et al., 1995), evaluation and characterization of germplasm (Bai et al., 2003; Mohapatra et al., 2003), verification of hybridity in interspecific crosses (Benedetti et al., 2000), tagging of genes (Martin et al., 1991) and marker-assisted selection (MAS) (Rahman, 2002; Sixin & Anderson, 2003) and gained importance due to its simplicity, efficiency and nonrequirement of sequence information (Karp et al., 1997). The objective of the present study was to determine the potential utility of RAPD technology for hybrid identification. The efficiency of the RAPD methodology in sorghum for seed purity testing was evaluated successfully, which would be beneficial for our sorghum breeding programs and seed industry. Material and Methods Seed Material: A total of two hybrid sorghum samples were received from Federal Seed Certification Department (FSCR&D), Islamabad, Pakistan. These were affixed as V1 and V2. Total Genomic DNA Isolation: Total Genomic DNA was isolated separately from ≈6 g of pre-soaked seed of two hybrid sorghum samples using a modification of the cetyltrimethyleammonium bromide (CTAB) procedure (Personal communication Mehboob-ur-Rahman). After RNase treatment, the DNA concentration was determined both by DyNA Quant 200 and by comparison with standard DNA, electrophoresed on a 0.8% agarose gel and the DNA was diluted in 0.1 X TE buffer to a concentration of 15 ng/ul for PCR analysis. PCR and Primers: For polymerase chain reaction (PCR), concentration of genomic DNA of two sorghum hybrids, 10 X PCR buffer without MgCl2, gelatin, MgCl2, dNTPs (dATP, dTTP, dGTP and dCTP), 10-mer random primer and Taq DNA polymerase were optimized. Ten-mer oligonucleotides were purchased from Operon Technologies Inc. Alameda, Calif. USA. Taq DNA polymerase together with 10 X PCR buffer, MgCl2 and dNTPs were purchased from MBI Fermentas, and gelatin was of Sigma grade. PCR amplification was carried out in a 25μL reaction volume containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 3 mM MgCl2, 0.1 mM each of dATP, dTTP, dGTP and dCTP, 30 ng of primer, 0.001% gelatin, 60 ng of genomic DNA and 0.2 unit of Taq DNA polymerase. A total of 20 random primers selected randomly from OPL and OPK series were used for PCR amplification. DNA amplification was performed using a thermal cycler programmed for first cycle of 5 min at 94 oC (initial strand separation); followed by 40 cycles of 1 min at 94 oC (denaturation), 1 min at 36 oC (annealing) and 2 min at 72 oC

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(primer extension) followed by one cycle of 10 min at 72 oC (final extension). The tubes were kept at 20 oC until removed. Analysis of RAPD Data: After amplification, PCR products were resolved on 1.2% agarose gel in 0.5 X Tris Borate EDTA (TBE) buffer, stained with ethidium bromide and photographed with Eagle Eye, gel documentation system. The amplified loci were scored as present/absent. The bivariate 1-0 data were used to estimate similarity on the basis of the number of shared amplification products (Nei & Li, 1979). Results and Discussion In the present study, RAPD analysis was applied to verify the identity of two sorghum hybrids. RAPD analysis has been successfully used for hybrid and parentage verification of other crop plants (Walker & Werner, 1997; Rahman et al., 2002; Asif et al., 2005). RAPD markers were also used to verify interspecific hybridization (Mei et al., 2004). However, reproducibility in amplification of RAPD markers is a major concern (Jones et al., 1998). In the present study, major/bright DNA fragments were scored to overcome the problem of reproducibility. Minor fragments, which are difficulty to repeat, were not scored because of random priming nature of the PCR reaction (Tessier et al., 1999). Genomic DNA of the two samples was surveyed with 20 random 10-mer primers. A total of 8 primers detected polymorphism (Fig. 1), while, 12 primers produced monomorphic pattern. A total of 121 bands were amplified, with an average of 6 bands per primer. Out of these amplified products, 33% were polymorphic. The level of polymorphism was varied with different primers. Ten fragments were amplified with each of OPL-10 and OPL-15 primers, and a minimum number of one band was amplified with OPL-3 and OPL-17. The mean number of amplified RAPD loci by each of the primer is relatively low compared to the earlier studies conducted on other crop species (Tao et al., 1993; Ayana et al., 2000) and is comparable with most of the previous reports in sorghum using RAPDs (Vierling et al., 1994; de Oliveira et al., 1996; Menkir et al., 1997; Agrama & Tuinstra, 2004). M

V1 V2

V1 V2

V1 V2

V1 V2

V1 V2 –Ve M

Arrow indicate polymorphic DNA locus Fig.1. Amplification profile of two sorghum hybrids with primer OPL-7; OPL-10; OPL14; OPK-19 & OPK-20. M=1kb ladder

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Table 1. Polymorphic primers along with sequences and number of loci amplified. Primer Sequence of the No. of loci Polymorphic % age of name primer loci polymorphism OPL-07 AGGCGGGAAC 8 2 25 OPL-08 AGCAGGTGGA 5 3 60 OPL-10 TGGGAGATGG 10 1 10 OPL-14 GTGACAGGCT 7 5 71.42 OPL-15 AAGAGAGGGG 10 1 10 OPK-12 TGGCCCTCAC 8 4 50 OPK-19 CACAGGCGGA 5 4 80 OPK-20 GTGTCGCGAG 9 6 66.67

The size of DNA fragments varied with different primers. The approximate size of the largest fragment amplified was in the range of 2.5 to 3.0 kb and the smallest easily recognizable fragment amplified was approximately 0.2 kb. In another study, a total of 213 fragments were amplified with 32 Operon primers from 22 sorghum genotypes and these fragments were in the range of 225 to 2600 bp in size (Agrama & Tuinstra, 2004). The findings of the present studies confirmed the efficiency of RAPD technique for verification of hybridity and purity identification. Because of the simple experimental procedures, the requirement of minimal amount of plant tissue and the possibility of automation (Terzi, 1997; Rahman et al., 2004), RAPD analysis would be very useful in breeding for rapid and early verification of hybridity in large seedling population and even purity testing of different seed lots, allowing the detection of true hybrids and verification of parentage of the hybrids and lines/cultivars. RAPD analysis has been successfully used for hybrid and parentage verification of other crop plants. In Cherokee rose, RAPD analysis allowed the identification of the erroneous classification of the hybrid ‘Silver Moon’ (Walker & Werner, 1997). RAPD markers were also used to verify interspecific hybridisation (Benedetti et al., 2000; Mei et al., 2004). In the present study, 78.98 % genetic similarity was assessed between the two sorghum hybrids. The extent of genetic diversity is high compared to cotton (Abdallah et al., 2001; Rahman et al., 2002). The high level of genetic diversity observed was probably associated with the extensive range of genetic diversity represented in the panel of sorghum genotypes (Agrama & Tuinstra, 2004). Such commonalities have been observed in soybeans, barley and corn (Powell et al., 1996; Russell et al., 1997; Pejic et al., 1998). It may be suggested that DNA-based markers such as RAPD have important implications for genotype identification and determination of hybridity and phylogenetic diversity of sorghum because of a requirement of simple agarose gel electrophoresis. RAPD analysis can be utilized efficiently in developing countries (Udupa et al., 1998; Agrama & Tuinstra, 2004). However, to have better understanding of genetic polymorphisms, it is imperative to survey sorghum genome with robust DNA marker tools; microsatellite analysis (SSR), amplified fragment length polymorphism (AFLP) and single nucleotide polymorphism (SNP) as has been achieved already for the human genome (Smith & Helentjaris, 1996). References Abdallah, A.M., O.U.K. Reddy, K.M. El-Zik and A.E. Pepper. 2001. Genetic diversity and relationships of diploid and tetraploid cotton revealed using AFLP. Theor. Appl. Genet., 102: 222-229.

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Agrama, H.A. and M.R. Tuinstra. 2004. Phylogenetic diversity and relationships among sorghum accession using SSRs and RAPDs. African J. Biotech., 2: 334-340. Asif, M., M. Rahman and Y. Zafar. 2005. DNA fingerprinting studies of some wheat (Triticum aestivum L.) genotypes using random amplified polymorphic DNA (RAPD) analysis. Pak. J. Bot., 37 (2): 271-277. Ayana, A., T. Bryngelsson and E. Bekele. 2000. Genetic variation of Ethiopian and Eritrean sorghum (Sorghum bicolor (L.) Moench) germplasm assessed by random amplified polymorphic DNA (RAPD). Gen. Res. Crop Evol., 47: 471-482. Bai, G., G. Peiguo and K. L. Frederic. 2003. Genetic relationships among head blight resistant cultivars of wheat assessed on the basis of molecular markers. Crop Sci. 43: 498-507. Barrett, B.A., K.K. Kidwell and P.N. Fox. 1998. Comparison of AFLP and pedigree-based genetic diversity assessment methods using wheat cultivars from the Pacific Northwest. Crop Sci,. 38: 1271-1278. Benedetti, D., G. Burchi, A. Mercuri, N. Pecchoni, P. Faccioli and T. Schiva. 2000. Random amplified polymorphic DNA (RAPD) analysis for the verification of hybridity in interspecific crosses of Alstroemeria. Plant Breed. 119: 443-445. Cao, W., G. Scoles, P. Hucl and R.N. Chibbar. 1999. The use of RAPD analysis to classify Triticum accessions. Theor. Appl. Genet., 98: 602-607. Colombo, C., G. Second, T.L. Valle and A. Charrier. 1998. Genetic diversity characterization of cassava cultivars (Manihot esculenta Crantz.) I. RAPD markers. Genet. Mol. Biol., 21: 69-84. de Oliveira, A.C., T. Richter, and J.L. Bennetzen. 1996. Regional and racial specifications in sorghum germplasm assessed with DNA markers. Genome 39: 579-587. De-Bustos, A., C. Soler and N. Jouve. 1999. Analysis by PCR-based markers using designed primers to study relationships between species of Hordeum (Poaceae). Genome, 42: 129-138. Devos, K.M. and M.D. Gale, 1992. The use of random amplified polymorphic DNA markers in wheat. Theor. Appl. Genet., 84: 567-572. Hashizume, T., P. Sato, M. Hirai. 1993. Determination of genetic purity of hybrid seed in watermelon (Citrullus lanatus) and tomato (Lycopersicon esculentum) using random amplified polymorphic DNA (RAPD). Jpn. J. Breed, 43:.367-375, 1993. Jones, C.J., K.J. Edwards, S. Castiglione, M.O. Winfield, F. Sala, C. Van-der-Weil, B. L. Vosman, M. Matthes, A. Daly, R. Brettschneider, P. Bettini, M. Buiatti, E. Maestri, N. Marmiroli, R.L. Aert, G. Volckaert, J. Rueda, A. Vazquez, and A. Karp. 1998. Reproducibility testing of RAPDs by a network of European laboratories. In: A. Karp, P.G. Isaac, and D.S Ingram (eds.) Molecular tools for screening biodiversity, Chapman & Hall, London, UK, pp 176-179. Karp, A., S. Kresovich, K.V. Bhat, W. G. Ayad and T. Hodgkin. 1997. Molecular tools in plant genetic resources conservation: a guide to the technologies; in IPGRI Technical Bulletin No. 2. International Plant Genetic Resources Institute, Rome, Italy. Martin, G. B., T. G. K. William and S. D. Tanksley. 1991. Rapid identification of markers linked to Pseudomones resistance genes in tomato by using random primers and near-isogenic lines. Proc. Natl. Acad. Sci. USA. 88: 2336-2340. McDonald, M.B. 1995. Genetic purity: from protein electrophoresis to RAPDs. In: Proceedings of the fiftieth annual corn & sorghum industry research conference. pp. 256-271. Mohapatra, T., S. Krishanpal, S. Singh, S. C. Swain, R. K. Sharma and N. K. Singh. 2003. STMSbased DNA fingerprints of the new plant type wheat lines. Curr. Sci. 84(8): 1125-1129. Klein-Lankhorst, R.M., A. Vermunt, R. Weide, T. Liharska, and P. Zabel. 1991. Isolation of molecular markers for tomato (2. Esculenta.) using random amplified polymorphic DNA (RAPD). Theor. Appl. Genet. 83: l08-l 14 Mei, M., N.H. Syed, W. Gao, P.M. Thaxton, C.W. Smith, D.M. Stelly and Z.J. Chen. 2004. Genetic mapping and QTL analysis of fiber-related traits in cotton (Gossypium)). Theor. Appl. Genet. 108: 280–291. Menkir, A., P. Goldsbrough and G. Ejeta. 1997. RAPD based assessment of genetic diversity in cultivated races of sorghum. Crop Sci. 37: 564-569. Mukhtar, M.S., M. Rahman and Y. Zafar. 2002. Assessment of genetic diversity among wheat (Triticum aestivum L.) cultivars from a range of localities across Pakistan using random amplified polymorphic DNA (RAPD) analysis. Euphytica, 128: 417-425.

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PAKISTAN BOTANICAL SOCIETY OFFICERS AND COUNCIL FOR 2006 President Dr. M. Ashraf Department of Botany, University of Agriculture, Faisalabad. Vice-President Dr. S.M. Saqlain Naqvi Department of Biochemistry, University of Arid Agriculture, Rawalpindi. Dr. Muhammad Tahir Rajput Department of Botany, University of Sindh, Jamshoro. Dr. Mufakhira Jan Durrani Department of Botany, University of Balochistan, Quetta. Dr. Farrukh Hussain Department of Botany, University of Peshawar, Peshawar. Mr. Shafiq-ur-Rehman Department of Botany, University of AJ&K Muzaffarabad. Secretary/Treasurer Dr. M. Yasin Ashraf NIAB, Faisalabad. Joint Secretary Dr. Syeda Saleha Hassney Department of Botany, University of Sindh, Jamshoro. Chief Editor Dr. Abdul Ghaffar, Department of Botany, University of Karachi.

COUNCIL The Officers of the Pakistan Botanical Society, the President and Secretary ex officio, Prof. Nuzhat Parveen, Shipowners College, Karachi; Dr. A. Qayyum Soomro, University of Sindh, Jamshoro; Dr. Shahida Hasnain, University of the Punjab, Lahore; Dr. Manzoor H. Soomro, PSF, Islamabad; Dr. Fazle Malik Sarim, University of Peshawar; Dr. A. Razzak Mahar, Shah Abdul Latif University, Khairpur; Dr. Tayyab Hussain Bukhari, Govt. College, Toba Tek Singh.

Vol. 38(5): Special Issue

Contents

December, 2006 Page Muhammad Ashraf and Habib-ur-Rehman Athar - Preface 1357 Sayed Anwar-ul-Haque - Salinity problems and crop production in coastal 1359 regions of Bangladesh - Review Article Abdallah Atia, Ahmed Debez, Mokded Rabhi, Habib-ur-Rehman Athar, 1367 and Chedly Abdelly - Alleviation of salt-induced seed dormancy in the perennial halophyte Crithmum maritimum L. (Apiaceae) Shahid Umar - Alleviating adverse effects of water stress on yield of sorghum, 1373 mustard and groundnut by potassium application M.U. Shirazi, M.A. Khan, Mukhtiar Ali, S. M. Mujtaba, S. Mumtaz, 1381 Muhammad Ali, B. Khanzada, M.A. Halo, M. Rafique, J. A. Shah, K.A. Jafri, and N. Depar - Growth performance and nutrient contents of some salt tolerant multipurpose tree species growing under saline environment Mumtaz Hussain, Muhammad Sajid Aqeel Ahmad and Abida Kausar - 1389 Effect of lead and chromium on growth, photosynthetic pigments and yield components in mash bean [Vigna mungo (L.) Hepper] Bechir Ben-Rouina, Chedlia Ben Ahmed, Habib-ur-Rehman Athar, and M. 1397 Boukhriss - Water relations, proline accumulation and photosynthetic activity in olive tree (Olea europaea L. cv “Chemlali”) in response to salt stress Ameer Khan, Muhammad Sajid Aqeel Ahmad, Habib-ur-Rehman Athar 1407 and Muhammad Ashraf - Interactive effect of foliar applied ascorbic acid and salt stress on wheat (Triticum aestivum L.) at seedling stage A. R. Gurmani, A. Bano and Muhammad Salim - Effect of growth regulators 1415 on growth, yield and ions accumulation of rice (Oryza sativa L.) under salt stress Muhammad Asif, Mehboob-ur-Rahman and Yusuf Zafar - Genotyping 1425 analysis of six maize (Zea mays L.) hybrids using DNA fingerprinting technology Sezgin Celik - Studies on the germination of three endangered Centurea species 1431 Vishandas, Zia-ul-Hassan, Muhammad Arshad and Ahmad Naqi Shah - 1439 Phosphorus fertigation at first irrigation due to its unavailability at sowing time prevents yield losses in Triticum aestivum L. M. Kashif Shahzad Sarwar, Ihsan Ullah, Mehboob-ur-Rahman, M. Yasin 1449 Ashraf and Yusuf Zafar - Glycinebetaine accumulation and its relation to yield and yield components in cotton genotypes grown under water deficit condition Muhammad Sajid Aqeel Ahmad, Qasim Ali, Rohina Bashir, Farrukh Javed 1457 And Ambreen Khadija Alvi - Time course changes in ionic composition and total soluble carbohydrates in two barley cultivars at seedling stage under salt stress Tayyaba Shaheen, Mehboob-ur-Rahman and Yusuf Zafar - Chloroplast rps8 1467 gene of cotton reveals the conserved nature through out plant taxa Chedlia Ben-Ahmed, B. Ben-Rouina, Habib-ur-Rehman Athar, M. 1477 Boukhriss - Olive tree (Olea europaea L. cv. “Chemlali”) under salt stress: water relations and ions content Jim Morris, John Collopy and Khalid Mahmood - Canopy conductance and 1485 water use in Eucalyptus plantations B. Shaharoona, Riffat Bibi, Muhammad Arshad, Zahir Ahmed Zahir, and Zia- 1491 ul-Hassan - 1-Aminocylopropane-1-carboxylate (ACC)-deaminase rhizobacteria extenuates ACC-induced classical triple response in etiolated pea seedlings Roubina Kauser, Habib-ur-Rehman Athar, and Muhammad Ashraf - 1501 Chlorophyll fluorescence can be used as a potential indicator for rapid assessment of water stress tolerance in canola (Brassica napus L.) Cont’d. inside back cover

Uzma Farooq and Asghari Bano - Effect of abscisic acid and chlorocholine chloride on nodulation and biochemical content of Vigna radiata L. under water stress Ruya Yilmaz, Serdal Sakcali, Celal Yarci, Ahmet Aksoy and Munir Ozturk - Use of Aesculus hippocastanum L. as a biomonitor of heavy metal pollution. Tariq Aziz, Rahmatullah, Muhammad Aamer Maqsood, Mukkram. A. Tahir, Iftikhar Ahmad and Mumtaz A. Cheema - Phosphorus utilization by six Brassica cultivars (Brassica juncea L.) from tri-calcium phosphate; a relatively insoluble P compound Gul Sanat Shah Khattak, Iqbal Saeed And Tila Muhammad - Breeding for heat tolerance in mungbean (Vigna radiata (L.) Wilczek) Nazila Azhar, M. Yasin Ashraf, M. Hussain and F. Hussain - Phytoextraction of lead (Pb) by EDTA application through sunflower (Helianthus annuus L.) cultivation: Seedling growth studies. Muhammad Ibrahim, Ambreen Anjum, Nabeela Khaliq, Muhammad Iqbal and Habib-ur-Rehman Athar - Four foliar applications of glycinebetaine did not alleviate adverse effects of salt stress on growth of sunflower Muhammad Aslam, Iftikhar A. Khan, Muhammad Saleem and Zulfiqar Ali - Assessment of water stress tolerance in different maize accessions at germination and at early growth stage Nudrat Aisha Akram, Muhammad Shahbaz, Habib-ur-Rehman Athar, and Muhammad Ashraf - Morpho-physiological responses of two differently adapted populations of Cynodon dactylon (L.) Pers. and Cenchrus ciliaris L. to salt stress M. Yasin Ashraf, Kalsoom Akhter, F. Hussain and Javed Iqbal - Screening of different accessions of three potential grass species from Cholistan desert for salt tolerance Nabila Tabbasam, Mehboob-ur-Rahman and Yusuf Zafar - DNA-based genotyping of sorghum hybrids Abdul Hameed, Muhammad Zaheer Ahmed and Muhammad Ajmal Khan Comparative effects of NaCl and seasalt on seed germination of coastal halophytes Tanwir Ahmad Malik, Sana-Ullah, and Samina Malik - Genetic linkage studies of drought tolerant and agronomic traits in cotton Qasim Ali, Habib-ur-Rehman Athar and Muhammad Ashraf - Influence of exogenously applied brassinosteroids on the mineral nutrient status of two wheat cultivars grown under saline conditions M. A. Khan, M.U. Shirazi, Mukhtiar Ali, S. Mumtaz, A. Sherin, and M. Y. Ashraf - Comparative performance of some wheat genotypes growing under saline water Naeem Iqbal and Muhammad Yasin Ashraf - Does seed treatment with glycinebetaine improve germination rate and seedling growth of sunflower (Helianthus annuus L.) under osmotic stress Irfan Afzal, Shahzad Maqsood Ahmad Basra, Amjad Hameed and Muhammad Farooq - Physiological enhancements for alleviation of salt stress in wheat Muhammad Saeed Akram, Qasim Ali, Habib-ur-Rehman Athar and Ahmed Saeed Bhatti - Ion uptake and distribution in Panicum antidotale Retz. under salt stress Sulman Shafeeq, Mehboob-ur-Rahman and Yusuf Zafar - Genetic variability of different wheat (Triticum aestivum L.) genotypes/cultivars under induced water stress

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Cont’d. on page 1770 Ihsan Ullah, Mehboob-ur-Rahman and Yusuf Zafar - Genotypic variation for 1679 drought tolerance in cotton (Gossypium hirsutum L.): seed cotton yield responses Ismat Nawaz, Zia-Ul-Hassan, Atta Muhammad Ranjha and Muhammad 1689 Arshad - Exploiting genotypic variation among fifteen maize genotypes of Pakistan for potassium uptake and use efficiency in solution culture Munir Ahmad, Zahid Akram, Muhammad Munir and Muhammad Rauf 1697 Physio-morphic response of wheat genotypes under rainfed conditions Qasim Ali, Habib-ur-Rehman Athar and Muhammad Ashraf - Ion transport 1703 in four canola cultivars as influenced by salt stress S.F. Afzali, M.A. Hajabbasi, H. Shariatmadari, K. Razmjoo, and A.H. 1709 Khoshgoftarmanesh - Comparative adverse effects of PEG- or NaCl-induced osmotic stress on germination and early seedling growth of a potential medicinal plant Matricaria chamomilla Mukkram Ali Tahir, Rahmatullah, Tariq Aziz, M.Ashraf, Shamsa Kanwal 1715 and Muhammad Aamer Maqsood - Beneficial effects of silicon in wheat (Triticum aestivum L.) under salinity stress Shamyla Nawazish, Mansoor Hameed and Shaista Naurin - Leaf anatomical 1723 adaptations of Cenchrus ciliaris l. from the salt range, Pakistan against drought stress Farah Naz, Shafqat Farooq, Rubina Arshad, Muhammad Afzaal and 1731 Muhammad Akram - Discriminating upland and lowland rice genotypes through proteomic approach Muhammad Akram, Shafqat Farooq, Muhammad Afzaal, Farah Naz, and 1739 Rubina Arshad - Chlorophyll fluorescence in different wheat genotypes grown under salt stress Muhammad Afzaal, Shafqat Farooq, Muhammad Akram, Farah. Naz, 1745 Rubina Arshad and Asghari Bano - Differences in agronomic and physiological performance of various wheat genotypes grown under saline conditions Rubina Arshad, Shafqat Farooq and Farooq-E-Azam - Rhizospheric bacterial 1751 diversity: Is it partly responsible for water deficiency tolerance in wheat? Shamaila Rafiq, Tahira Iqbal, Amjad Hameed, Zulfiqar Ali Rafiqi and 1759 Naila Rafiq - Morphobiochemical analysis of salinity stress response of wheat Published by the Pakistan Botanical Society. Printed at Karachi University Press, Karachi-75270, Pakistan. Phone: 9243186, 4969237. Copies available from the Chief Editor, Pakistan Journal of Botany, Department of Botany, University of Karachi, Karachi-75270, Pakistan. Phone: 4387867. Price: Each issue Rs. 100/ US$20; Vol. Rs. 600/US$120 or in exchange.