INTERNATIONAL JOURNAL OF AGRICULTURE & BIOLOGY ISSN Print: 1560–8530; ISSN Online: 1814–9596 13–1174/2014/16–6–1098–1104 http://www.fspublishers.org Full Length Article To cite this paper: Habib, I., M. Rauf, J. Qureshi, M. Ahmed, S. Mansoor and N.A. Saeed, 2014. Optimization of somatic embryogenesis and Agrobacterium- mediated transformation of elite wheat (Triticum aestivum L.) cultivars of Pakistan. Int. J. Agric. Biol., 16: 1098−1104 Optimization of Somatic Embryogenesis and Agrobacterium-mediated Transformation of Elite Wheat (Triticum aestivum) Cultivars of Pakistan Imran Habib*, Muhammad Rauf, Javed Qureshi, Moddassir Ahmed, Shahid Mansoor and Nasir Ahmad Saeed National Institute for Biotechnology and Genetic Engineering (NIBGE), Faisalabad, Pakistan *For correspondence: imranuaf@hotmail.com Abstract Immature embryos of twenty six wheat genotypes were regenerated on Murashige and Skoog (MS) medium supplemented with different levels of plant growth regulator (PGRs). 2, 4-D at 2 mg L -1 level was found to be the most favorable callus inducer. Bobwhite exhibited maximum embryogenic callus production followed by ‘Ufaq-2000’, ‘Punjab-2011’ and ‘Sehar- 2006’. Regeneration potential of selected wheat genotypes was evaluated on two PGRs indole-3-acetic acid (IAA) and kinetin (N6-furfuryladenine) in 4 different combinations. The auxin-cytokinin combination in IK4 (1 mg L -1 IAA and 1 mg L -1 Kinetin) media was the most promising for regeneration as variety Bobwhite presented the highest plantlet frequency. Immature embryos of cultivar ‘Sehar-2006’ were utilized for Agrobacterium mediated transformation and GUS activity was observed in callus to determine the expression pattern. Transgenic plants of ’Sehar-2006’ were confirmed by PCR and Southern blotting. Transformation efficiency of 2.45 % was achieved which reinstated the viability of optimized regeneration system and this particular wheat genotype for utilization in crop improvement program. © 2014 Friends Science Publishers Keywords: Agrobacterium; Callus; IAA; Kinetin; Regeneration, Transformation, Wheat Introduction Employing state of the art crop improvement techniques like biotechnology and genetic engineering is promising. Emphasis has been shifted for deployment of genetic modification systems in modern day crops for rapid and sustainable crop improvement. Plant tissue culture is the foremost tool in any plant transformation system (Debnath et al., 2006; Noor et al., 2009). Despite all the efforts, most cereal crop in particular, wheat has proven to be very tedious for regeneration of embryogenic callus (Fazali- nasab et al., 2012; Bouiamrine et al., 2012). The choice of regenerable and highly transformation efficient genetic material is very limited and confined to only a few specialized varieties like bobwhite. One way to overcome these bottle necks is to screen and utilize our indigenously adaptive and high yielding wheat cultivars for the purpose of exploring their regeneration potential and transformation efficiency. This will reduce the development period of a desirable transgenic material. Moreover, the exact concentration and combination of these auxins and cytokinins in the nutrient media are critical and play a decisive role in regeneration initiation particularly in crops like wheat (Saad et al., 2004; Zarif et al., 2013). This study describes a highly efficient callus induction, embryogenic callus production and plant regeneration system for local elite wheat cultivars. Immature embryos were utilized as explant and cultured on different levels and combinations of phytohormons to optimize the PGR concentrations and to establish best responsive wheat genotypes for Agrobacterium mediated transformation. This optimized high frequency callus induction and regeneration system will therefore provide ample embryogenic material for plant transformation and higher rates of plantlet regeneration for better transformation efficiency of local and exotic wheat genotypes. Materials and Methods Plant Material and Explant Preparation Twenty six wheat genotypes including twelve commercial cultivars (‘TD-1’, ‘Uqab-2000’, ‘SH-2002’, ‘AS-2002’, ‘GA-2002’, ‘GD-2002’, ‘Bhakkar-2002’, ’Sehar-2006’, ‘Shafaq-2006’, ‘Inquilab-91’, ‘Lasani-2008’, ‘Punjab- 2011’), seven old varieties (‘Punjab-76’, ‘Chakwal-86’, ‘Pak-81’, ‘Watan’, ‘Blue Silver’, ‘TW-471’ and ‘Ufaq- 2000’), six advanced breeding lines (‘V-03079’, ‘V-04188’, ‘Millat-2011’, ‘V-07076’, ‘Galaxy-2013’ and ‘G-98-4’) and one exotic highly regenerable check ‘Bobwhite’ were evaluated for regeneration potential. After 14-16 days post anthesis freshly removed seeds were surface sterilized with 70% ethanol for one min. After rinsing with sterilized distilled water, these seeds were washed with 20% Clorox bleach (5.25% Sodium hypochlorite) and 0.1% Tween 20 for 10 min. Seeds were thoroughly rinsed three times with sterile distilled water and excess water was removed by placing the seeds on sterile filter paper under aseptic conditions.