The citrus fruit ranks at the top in production and trade among all fruits produced the world over. It is highly prized and remunerative fruit, cultivated almost all over the world. Citrus comprises about 40 percent of all fruits that grow in Pakistan. It is cultivated over an area of 206,569 hectares with an average annual production of about 2.36 million tons per annum. Pakistan citrus industry is facing different pre-harvest and post-harvest problems including bacterial, viral diseases, pest attack, poor management and under developed citrus industry. All the mentioned problems lead to failure and declining citrus industry. The current decline in citrus production in the country is attributed to a great extent to Huanglongbing (HLB) or Citrus greening disease. HLB is transmitted by grafting and psyllids Diaphorina citri. HLB disease is hard to control due to having an aggregated nature of disease symptoms, prolong incubation period and may remain symptomless in certain host plants. The movement of HLB bacterium is unevenly distributed in phloem sieve tubes, leaf midrib, and vascular tissues in bark, floral parts and in roots. These symptomless infected plants prove more dangerous as they are continuous source of infection spread through the vector. Another difficulty related to this disease is its symptoms as these are similar with nutrient deficiencies or other viral diseases such as CTV in citrus plants. This study was consummated to characterize the causative agent of citrus HLB disease in different orchards of Pakistan. The distribution of HLB disease across agroecological zones and citrus varieties of two orchards viz. “Citrus Research Institute, Sargodha (CRI)” and “Bursha Citrus Research & Development Center, Sillanwali, Pakistan” were observed. Symptomatic and asymptomatic leaves of citrus trees were randomly sampled, scored and stored at 4°C. Bacterial DNA isolated from the leaf midribs was subjected to PCR to amplify specific ribosomal regions on Candidatus Liberibacter genome (16S rDNA, 16S/23S rRNA) and OMP gene fragments. These amplicons were sequenced and systematically analyzed for homology. The data from CRI orchard’s samples revealed HLB infection in symptomatic as well as non-symptomatic trees of all varieties. All the six groups of citrus were infected with HLB infectious agent. Kamquat and Sweet Oranges were severely infected with HLB associated bacterium having 86% and 66% prevalence. Grapefruit revealed Candidatus Liberibacter asiaticus (Las) incidence in 40% samples. Lemon and limes revealed 47% and Mandarin showed 31% of HLB infection rate. Of the total 466 citrus samples of 6 groups, 249 samples were found positive for HLB pathogenic bacterium. Total 53% samples of CRI were infected with Candidatus Liberibacter asiaticus. The varietal samples collected from Sillanwali orchard revealed 11% HLB infection, of which only 4 varieties had harboured Candidatus Liberibacter asiaticus causing HLB disease whereas 34 varieties were found HLB disease free. The current study was conducted to develop Agrobacterium-mediated transformation protocol of epicotyls explants of rough lemon (rootstock) using pAFS-SUC2-D4E1 recombinant vector. Transformation experiments were performed with Agrobacterium tumefaciens strains LBA4404 and GV3101 harboring a binary vector pCAMBIA 1301. Kanamycin, Acetosyringone concentrations, optical density of Agrobacterium culture, preculturing and co-culturing period were investigated during transformation. Transgenic shoots were selected on 100 (mg/L) kanamycin along with (250 mg/L) each of cefotaxime and vancomycin for effective inhibition of Agrobacterium growth. The Murashige and Skoog (MS) medium containing 200 μM acetoseryngone proved to be the best inoculation and co-cultivation medium for transformation. MS medium supplemented with 3 (mg/L) of 6-benzylaminopurine (BAP) showed maximum regeneration efficiency of the transformed explants. By combining the best combinations of the transformation factors, achieved highest transformation efficiency (24.19 %). The integration of SUC2-D4E1 expression cassette in transgenic plantlets was confirmed by conventional PCR analysis and mRNA of the gene in the host plant. A rapid and efficient protocol was established to produce HLB resistance against the infectious agent in transgenic plants. Although the transformation efficiency was not very high (24.19 %) which can be improved with further developments, however, this would provide an effective preliminary procedure for Agrobacterium mediated transformation in citrus. The protocol opens up new avenue for genetic improvement of the current as well as other citrus cultivars with valuable genes to attain sustainable and higher production.