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INTEGRATION OF NOVEL CHLOROPHYLL GENES FROM BLACK PINE INTO THE CHLOROPLAST GENOME OF TOBACCO

SHAHID NAZIR¹ AND MUHAMMAD SARWAR KHAN^2*

Abstract: Gymnosperms owing the presence of dark-operative protochlorophyllide oxidoredcuatse pathway are able to reduce protochlorophyllide to chlorophyllide leading to the formation of chlorophyll in dark, whereas angiosperms are unable to do this and are photosynthetically less efficient. Plastid encoded genes chlL and chlN are reported to be involved in the functioning of the enzyme Dark-operative Protochlorophyllide Oxidoreductase (DPOR). The genes were isolated from black pine and cloned into the species-specific chloroplast targeting vector; harboring FLARE-S, a fluorescent selection marker. The final transformation vector was used to introduce genes into the tobacco chloroplast genome using biolistic approach. Here, we report stable integration of both genes along with translationally fused marker genes (gfp and aadA) into the inverted repeat region of plastome which was confirmed by Polymerase Chain Reaction and Southern blot analysis. Morphological and physiological analyses of the transgenic plants compared with non-transformed wild type tobacco plants revealed that the activation of dark-operative pathway requires additional factors/genes to chlL and chlN genes to develop chlorophyll, and consequently photosynthetically competent chloroplasts.

^1,2National Institute for Biotechnology and Genetic Engineering (NIBGE), P.O. Box 577, Faisalabad, Pakistan

²Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture, Faisalabad Pakistan

^*Corresponding: sarwarkhan_40@hotmail.com