Several approaches of genetic manipulation have been performed to increase carotenoid levels in different plant species and tissues. Over the past decade many of the genes encoding the enzymes of the carotenoid biosynthetic pathway such as isopentenyl diphosphate/dimethylallyl diphosphate synthase isomerase, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase, ζ-carotene desaturase, lycopene α- and β-cyclase, β-carotene hydroxylase, neoxanthin synthase and even the subsequent epoxidase have been cloned from both plant and microbial sources (Herbers, 2003; Cunningham and Gantt, 1998; Hirschberg, 2001; Fraser and Bramley, 2004; Al-Babili et al., 2000). Previous reports describe modifications of the amounts and types of carotenoid that accumulate in experimental and food plants by transgenic manipulation of the carotenoid biosynthetic pathway (Fraser and Bramley, 2004). The over-expression of phytoene synthase has a particularly potent effect on storage organ carotenoid levels and results in increases in total carotenoid content in carrot roots (Hauptmann et al., 1997), tomato fruit (Fraser et al., 2002), canola seed (Shewmaker et al., 1999), Arabidopsis seed (Lindgren et al., 2003) and potato tubers (Ducreux et al., 2005). The extent of the increase varied between 1.6 fold in tomato fruit, 50 fold in canola seed up to 6 fold in potato tubers. Transgenic manipulation of potato tubers has been used successfully to elevate carotenoid levels and increase the spectrum of carotenoids that accumulate to significant levels (Romer et al., 2002; Ducreux et al., 2005). In recent work of Diretto et al. (2007b), expression of three Erwinia genes encoding phytoene synthase (CrtB), phytoene desaturase/carotene isomerase (CrtI) and
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lycopene beta-cyclase (CrtY), under the control of a tuber specific promoter, demonstrated the conversion of geranylgeranyl diphosphate (GGPP) into β-carotene. This resulted in tubers with a deep yellow (golden) phenotype without any adverse leaf phenotypes. Total carotenoids increase up to 20-fold (114 µg/g dry weight) and β-carotene up to 3600-fold (47 µg/g dry weight). This golden potato is the highest carotenoid and β-carotene content reported for biofortified potato as well as for any of the four major staple foods. Consuming 250 gm of this tuber flesh is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A. This consumption (47 µg/g dry weight) as stated by Diretto et al. (2007b), is even better than Golden Rice 2 with 31 µg/g dry weight of β- carotene.
Meanwhile silencing the first step in the epsilon-beta branch, LCYe, increases total carotenoids up to 2.5 fold and β-carotene up to 14 fold (Diretto et al, 2006), whereas silencing the non-heme β-carotene hydroxylases CHY1 and CHY2 in the tuber showed more dramatic changes with total carotenoids increasing up to 4.5 fold and β-carotene up to 38 fold (Diretto et al, 2007a). However, zeaxanthin levels decreased, whereas neoxanthin and violaxanthin stayed the same. CRTISO, LCYb and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species (Diretto et al, 2007a). Romer et al. (2002) reported that down-regulation of zeaxanthin epoxidase in tubers of S. tuberosum led, in some transgenic lines, to a dramatic increase in the zeaxanthin content and the total tuber carotenoid content up to 5.7 fold. In this case the conversion of zeaxanthin to violaxanthin was inhibited leading to accumulation and elevation of zeaxanthin up to 4 to 130 fold. The values reaching up to 40 µg/g dry weight depending on the transgenic lines and tuber development. Ducreux et al. (2005) revealed that expression of a bacterial crtB gene encoding phytoene synthase led to
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6-fold higher carotenoid levels with violaxanthin, antheroxanthin, lutein and β-carotene were the major carotenoids that accumulated in the transgenics but only lutein levels increase with the increase of β-carotene and total carotenoids. Morris et al. (2006) found that over expression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase (DXS) gene in potato tubers resulting 2 fold increases in total carotenoid and 6-7 fold increase in phytoene. In all cases, metabolic engineering of potato has led to the production of more β- carotene, phytoene, lutein and zeaxanthin (Romer et al. 2000; Fraser et al. 2002; Ducreux et al. 2005; Diretto et al, 2007a; 2007b), and the accumulation of astaxanthin, a new and high-economic value carotenoid, in potato tubers (Gerjets and Sandmann 2006; Morris et al., 2006b).
Rather than directly regulating carotenoid biosynthesis, recent work by Lu et al. (2006) through transformation of the Or gene into wild type cauliflower converts the white color of curd tissue into distinct orange colour with increased levels of β-carotene. Or
gene, which encodes a DnaJ cysteine-rich domain-containing protein, leads to the formation of large membranous chromoplasts in the cauliflower curd cells which strongly associated with carotenoid accumulation (Li et al. 2001; Lu et al. 2006). Similarly when the Or gene under the control of a potato granule-bound starch synthase (GBSS) promoter was introduced into a potato (Li and Van Eck, 2007), the orange-yellow flesh tubers were produced. The total carotenoid levels were 6 fold higher than the non-transformed and control. These orange-yellow flesh tubers is due to the extra orange bodies structures in chromoplasts, which provide a metabolic sink to facilitate accumulation of carotenoids, whereas the tubers in the non-transformed and vector-only controls contain exclusively various sizes of starch grains in amyloplasts. Although previous studies have shown that overexpression of genes in the carotenoid biosynthetic pathway resulted increased levels of
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carotenoids, modification of sink capacity also proven as a new strategy to enhance carotenoids in storage tissues of food crops. Manipulation of both tools perhaps can be more effective to enhance carotenoids level quantitatively and qualitatively in order to meet the requirement for human nutrition and health. Hence, genetic engineering improvement of carotenoid content in potato tubers urgently requires more detailed knowledge of the diversity of carotenoid pigments in potatoes and environmental factors influencing their accumulation to better understand tubers carotenogenesis regulation. In order for this to be achieved, it is important to identify the best cultivars for targeting specific genetic manipulations and to understand the key control factors for carotenoid accumulation in potato tubers.