RESEARCH ARTICLES CURRENT SCIENCE, VOL. 105, NO. 1, 10 JULY 2013 72 *For correspondence. (e-mail: manamohan.s.m@gmail.com) Anthocyanin enrichment of tomato (Solanum lycopersicum L.) fruit by metabolic engineering Manamohan Maligeppagol 1, *, G. Sharath Chandra 1 , Prakash M. Navale 1 , H. Deepa 1 , P. R. Rajeev 1 , R. Asokan 1 , K. Prasad Babu 1 , C. S. Bujji Babu 2 , V. Keshava Rao 2 and N. K. Krishna Kumar 3 1 Division of Biotechnology, 2 Division of Plant Physiology and Biochemistry, 3 Division of Entomology and Nematology, Indian Institute of Horticultural Research, Hesaraghatta Lake (PO), Bangalore 560 089, India Transgenic tomato plants accumulating high amounts (70–100 fold) of anthocyanin in the fruit were deve- loped by the fruit specific expression of two transcrip- tion factors, Delila and Rosea1 isolated from Antirrhinum majus. The transgenic tomato plants were identical to the control plants, except for the accumu- lation of high levels of anthocyanin pigments through- out the fruit during maturity, thus giving the fruit a purplish colour. The total carotenoids, including lyco- pene levels were unaffected in the anthocyanin-rich fruits, while its antioxidant capacity was elevated. The gene expression analysis confirmed the elevated ex- pression of the downstream genes of the anthocyanin pathway due to the expression of the transcription factors and the expression levels coincided with the fruit ripening stages, highest expression occurring during the breaker stage. Anthocyanin-rich tomato fruit is important in view of the protective function of these compounds on consumption against a number of lifestyle-related diseases. Keywords: Anthocyanin, carotenoids, metabolic engi- neering, transgenic tomato. DIETARY anthocyanins are potent antioxidants, and modi- fiers of cell signalling pathways and their consumption is known to confer protection against cardiovascular dis- eases (CVDs), cancers, diabetes, arthritis, etc. 1,2 . They belong to flavonoids, a large group of polyphenolic com- pounds abundant in some fruits and vegetables, such as pomegranate, Indian black berry or jamun (Syzygium cumini L.), red/blue grapes, purple corn, purple cabbage, etc. The World Health Organization (WHO) estimates that 80% CVDs and one-third of cancers can be avoided by healthier diet (WHO Fact Sheet, Global Strategy on Diet, Physical Activity and Health). Anthocyanins are one such group of nutraceuticals known to play a protec- tive function against chronic diseases. Increased con- sumer awareness of the protective functions of health foods and higher incidence of CVDs and cancers in the population has increased the demand for health foods, including the anthocyanin-rich fruits and vegetables. However, anthocyanins are not present in the edible parts of some important crop plants, such as tomato, which is one of the most consumed vegetables world- wide. Naturally tomato fruits accumulate only traces of anthocyanins mainly in the peel 3 and small amounts of other flavonoids, such as naringenin chalcone and fla- vonols in the fruit 4,5 . Tomato is an ideal candidate for anthocyanin enrich- ment due to its widespread regular dietary intake in fresh, cooked and processed forms. Higher agronomic produc- tivity, year-round availability and amenability for genetic engineering, coupled with consumption pattern make tomato a suitable crop for anthocyanin enrichment 6 . Attempts have been made to breed tomato fruits with en- hanced anthocyanin content by classical plant breeding 7–10 . The genetic characterization of these plants revealed that the alleles, Aubergine (Abg), Anthocyanin fruit (Aft) and atroviolacea (atv) exhibited varying levels of anthocyanin accumulation in the epidermis only but not throughout the fruit; also the fruit size was negatively correlated with anthocyanin content. Tomato fruits with significantly higher levels of carotenoids and flavonoids were obtained by the fruit-specific RNA interference-mediated suppres- sion of an endogenous photomorphogenesis-related regu- latory gene DE-ETIOLATED 1 (DET1), demonstrating that the regulatory genes could be effectively employed in metabolic engineering to enhance the levels of a wide range of phytonutrients 11 . Manipulation of transcription factors is an ideal strategy to modify the expression of multiple target genes. In tomato, overexpression of Anthocyanin 1 (Ant1), a transcription factor regulating anthocyanin production has led to the accumulation of anthocyanins in fruit skin and a layer immediately below it 12 . Similarly, two transcription factors Leaf Color (LC) and Colorless1 (C1) from maize were introduced into to- mato to induce anthocyanin accumulation in fruits 13 , however no anthocyanin accumulation could be observed,