With globally increasing demand for flowers with novel colours has created huge demand for rare and unusual colours in ornamental crops and the result of that every day novel varieties are being added to the market. But the conventional techniques of creating variability has certain limitation and to overcome this problem, genetic engineering is a much needed tool through which we can broaden the gene pool. As its well known fact that flavonoids, carotenoids and betalains are major floral pigments responsible for expression of colours in flower. Between these three groups, the flavonoids contribute most to the range and type of coloured pigments in plants and that's why flavonoid pathway has primarily targeted in genetic engineering techniques as a means to alter flower colour. Flavonoids consist of more than 10 classes of compounds. Anthocyanins confer orange, red, magenta, violet and blue colours. Aurones and chalcones are yellow pigments while flavones and flavonols are colourless or very pale yellow.

Final visible colour in those species in which colour is primarily derived from anthocyanins and hundreds of anthocyanins have been reported [Veitch and Grayer, 2008], they are primarily based upon six common anthocyanidins (chromophores of anthocyanins); pelargonidin, cyanidin, peonidin, delphinidin, petunidin and malvidin. In terms of biosynthesis, since peonidin is derived from cyaniding and petunidin and malvidin are both derived from delphinidin, there are only three major anthocyanidins; pelargonidin, cyanidin and delphinidin. Blue flowers tend to have delphinidin and its derivatives and intense red flowers tend to have pelargonidin as the anthocyanidin base. An increase in the number of hydroxyl groups on the B-ring imparts a bluer colour to the anthocyanins derived from the anthocyanidin, while methylation of the 3' or 5'-hydroxyl group results in a slight reddening. The expression of anthocyanins is a result of combination of various factors, such as anthocyanin structure, type and concentration, co-existing compounds (co-pigments), metal ion type and concentration, pH of vacuoles, anthocyanin localization and shapes of surf ace cells all contribute to final flower colour [Yoshida et al. 2009]. Through combinations of these factors, plant species have evolved flower colours to attract insect pollinators. Each of these factors is regulated by a number of genes, many of which now have been cloned. Now blockage of CHS is possible which results in to flavonoid-free transgenic plants but these flavonoids have been found to play an important role in UV protection, general plant defence and signalling regulation of the CHS gene may not represent an ideal strategy to develop novel varieties ( Winkel-Shirley, 2002). Therefore, other methods need to be targeted. The few successful examples of colour modification are given below.

Transgenic carnations:
The modification of flower colour via genetic engineering has generally focused on metabolic engineering of the flavonoid pathway. Commercialisation of genetically engineered flowers is currently confined to carnations only and its first flower crops which GM varieties were grown commercially. The down regulated carnation F3H, anthocyanin levels were not the only change observed in the transgenic carnations produced. The carnation were also more fragrant due to an increase in methylbenzoate, which may be considered a more positive outcome when commercialising these flowers (Zuker et al., 2002). The blue coloured 'moon series' of carnation is grown in Ecuador, Colombia and Australia, and there is now a ten year history of safe use which was produced by over-expression of a petunia F3'5'H gene under the control of a constitutive promoter in a pelargonidin producing carnation variety produces petals in which delphinidin derivatives contribute to about 70% of total anthocyanins [Holton and Tanaka, 1994]. However, there was only a slight colour change toward blue. Another variety 'Moondust' were developed through expression of petunia F3'5'H (under the control of a promoter region from the snapdragon CHS gene) and petunia DFR (under the control of a constitutive promoter) genes, resulted in exclusive accumulation of delphinidin derivatives and significant colour change toward blue. The variety 'Moonshadow' with dark violet colour flowers was developed through expression of a pansy F3'5'H gene (under the control of a promoter region from the snapdragon CHS gene) and a petunia DFR-A gene (under the control of its own promoter and terminator regions) resulted in transgenic plants which also exclusively accumulated delphinidin but at a higher concentration.

Transgenic rose:
The rose (Rosa hybrida) is most popular and top ranked cut flower in market with large number of varieties originated from diverse parents. But the demand for novel and unique rose varieties in market is ever increasing and that's reason rose breeders are always under pressure to cater the demand of novelty in market. The present day cultivated roses are the product of extensive inter-specific hybridization utilising almost all known wild species which resulted into varieties with all colours with wide range of hues. However, despite the huge range of flower colours that have been bred, rose lack any varieties in the bluish range of flower colour because the genus Rosa does not have the biochemical pathway leading to delphinidin-based anthocyanins. Therefore, the journey began for development of true blue coloured roses through gene engineering techniques. Genes and biochemical pathway of anthocyanins from many blue coloured flowers have been studied and some suitable genes and are incorporated to get the blue colour. But rose cultivars that have higher vacuolar pH, large amount of flavonols (co-pigments) and weak or no F3'H activity were selected in order to enhance the blue hue of the transgenic petals and to achieve high content of delphinidin. Expression of a F3'5'H gene from petunia, gentian or butterfly pea in rose resulted in no or little delphinidin accumulation in the petals of transgenic plants, even though these genes were shown to be functional in petunia, carnation or yeast. In contrast, expression of pansy (Viola spp) F3'5'H genes in rose resulted in a significant amount of delphinidin derived anthocyanins accumulating in petals of the transgenic plants [Brugliera et al. 2004]. Rose cultivars with higher vacuolar pH, large amount of flavonols (co-pigments) and weak or no F3'H activity should be selected in order to enhance the blue hue of the transgenic petals and to achieve high content of delphinidin. However Brugliera et al., (2004) used such cultivars for expression of pansy F3'5'H genes resulted in transgenic lines in which 95% of the anthocyanidins was delphinidin. The colour of the flowers in these lines were of a significantly bluer hue than any conventionally bred cultivar.

Mizutani et al., (2003) Evaluation of Post Transcriptional gene silencing methods using flower color as the indicator. Plant Cell Physiol. 44:122.
Winkel-Shirely B (2002). Biosynthesis of Flavonoids and effects of stress. Curr. Opin. Plant Biol. 5: 218-223.
Zuker et al., (2002) Modification of flower colour and fragrance by antisense suppression of the flavanone 3-hydroylase gene. Mo. Breed. 9: 33-41.
Yoshida, K.; Mori, M.; Kondo, T. (2009) Blue flower color development by anthocyanins: From chemical structure to cell physiology. Nat. Prod. Rep., 26: 884-915.
Holton, T.A.; Tanaka, Y. (1994) Transgenic flowering plants. Patent Publication Number WO/94/28140.
Brugliera, F.; Tanaka, Y.; Mason, J. (2004) Flavonoid 3'5'-hydroxylase gene sequences and uses therefor. Patent Publication Number US7612257, March 11.
Veitch, N.C.; Grayer, R.J. (2008) Flavonoids and their glycosides, including anthocyanins. Nat. Prod. Rep., 25:555-611.
Tanaka, Y., Brugliera, F. and Chandler, S. (2009) Recent Progress of Flower Colour Modification by Biotechnology Int. J. Mol. Sci., 10: 5350-5369

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