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TRANSFORMATION OF TOMATO (LYCOPERSICON ESCULENTUM MILL.) WITH ARABIDOPSIS EARLY FLOWERING GENE APETALAI (API) THROUGH AGROBACTERIUM INFILTRATION OF RIPENED FRUITS

M. HASAN¹, A. J. KHAN², S. KHAN³, A.H.SHAH⁴, A. R. KHAN⁴ AND B. MIRZA⁵

Abstract: In order to develop an easy, rapid and efficient protocol for the genetic transformation of tomato (Lycopersicon esculentum Mill.), without involving tissue culturing, ripened fruits of tomato were infiltrated with Agrobacterium harboring plasmid pROKII GUSint AP1. The plasmid contains Arabidopsis early flowering gene AP1, GUS reporter gene and NPTII as a selectable marker gene. Both transient and stable GUS expressions were studied with different parameters such as various incubation periods (48, 72 and 96 hours) and tissue type effect. The effects of infiltration on germination of seeds both on selection and non-selection media were studied. The percentage of germination of seeds on non-selection medium was 14% lesser than control, while the germination percentage on selection medium was significantly higher for treated seeds (65-74%) as compared to control (5%), with 48 hours incubation period having maximum (74%). Among kanamycin resistant plants 94.9% to 87.9% showed stable GUS expression, while 5.1% to 12.07% were escapes. Transient GUS expression exceeded stable GUS expression. Different tissues of the same plant showed variable stable GUS expression (data not shown here). The effect of incubation period was highly significant (p<0.05), with 48 hours incubation period having maximum efficiency (68%). The effect of tissue type on both transient and stable GUS expression was also prominent. Overall, high transformation efficiency 68% to 54% was obtained in seedlings raised from seeds collected from infiltrated fruits. Transformation was confirmed by analyzing the PCR amplified product of AP1, GUS and NPTII genes. This depicted a novel method of transformation in plants in general and tomato in particular.

¹G.P.G.C. Bannu
²Gomal Centre of Biotechnology and Biochemistry, Gomal University, D.I. Khan, Pakistan

³Kohat University of Science and Technology, Kohat.

⁴Department of Biological Sciences, Gomal University, D. I. Khan,

⁵Faculty of Biological Sciences, Quaid-e-Azam University Islamabad, Pakistan.