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2.21.2 Culture Conditions

Carthamus tinctorius is an oilseed crop cultivated commercially in Iran (Amini et al. 2013), and its seeds are used for their edible oil (Tayefi-Nasrabadi et al. 2011). This crop plant is rich source of fatty acids (Knowles 1989). In vitro selection technique has been used to improve abiotic environmental stresses such as cold hardiness, salt tolerance, and drought tolerance (Zair et al. 2003; Bajji et al. 2004; Gawande et al. 2005). Recent progress in the genetic manipulation of plant cells through tissue culture has opened new possibilities in crop improvement in which crop species can be improved without interfering with a large portion of the genes and without introducing alien or exogenous genetic material (Hassan et al. 2004; Farshadfar et al. 2012; Mahmood et al. 2012; Shabani et al. 2013).

      Carthamin is stable to heat and light and is used in baked goods and beverages. The callus was obtained from flower bud explants and could also be put into suspension. The production of carthamin was enhanced by selecting with components of various media as well as by feeding of precursors. It was also found that addition of cellulose, chitin, or chitosan increased production of the carthamin pigment. Supplementation of D-phenylalanine and removal of Mg alone or both Mg and Ca from the culture medium also enhanced the production of carthamin. C. tinctorius L. is commonly known as safflower. C. tinctorius extracts and oil are important in development of drugs with numerous pharmacological activities in the world. Carthamidin, isocarthamidin, hydroxysafflor yellow A, safflor yellow A, safflamin C, and luteolin are the main constituents reported from this plant. Caryophyllene, p-allyltoluene, 1-acetoxytetralin, and heneicosane were identified as the major components from C. tinctorius flower (Asgarpanah and Kazemivash 2013).

      Application of lower concentration of berberine inhibited growth of the callus cultures of C. tinctorius, but the high concentration of berberine tended to stimulate the callus growth of Coptis japonica var. japonica. Among callus cultures of the five species described above, 4-desoxypyridoxine inhibited growth of the callus cultures of C. tinctorius (Yamamoto 1980).

      Phenylalanine (¹⁴C) was administered to the cell cultures and the in vivo flowers of C. tinctorius, and the biosynthetic activity of carthamin in these two materials was compared. The cultured cells took up positively the fed substrate, but they could not incorporate the label into carthamin, while incorporation of the radioactivity from phenylalanine into the red pigment occurred in the intact flowers. Polyphenol-oxidizing enzymes were operative normally in the mother explant, whereas their activity patterns changed altogether in the cultured cells, where kurenamin, a new reddish pigment, is produced actively (Saito et al. 1988, 1993).

The maximum percentage of callogenesis from leaf explants was obtained in MS medium supplemented with 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP), and internodal explants also responded to callus formation in MS medium containing NAA and BAP under light condition. Furthermore, MS medium supplemented with 2, 3, 5-triiodobenzoic acid (TIBA) proved to be the best for callus induction and proliferation from root explants under dark condition. Under light condition, leaf and intermodal explant-derived calli were best proliferated; however dark condition had more impact on callus induction as compared to light condition for root explants (Karakaya et al. 2004; Ali and Afrasiab 2014).

      Callus regeneration systems for C. tinctorius were evaluated by using root, hypocotyl, cotyledon, and leaf explants, and the influence on organogenesis of seedling age, media factors, growth regulators, and excision orientation was studied. Supplementation of the medium with various concentrations of indole-3-acetic acid (IAA), NAA, BAP, and Kinetin in the MS medium was found effective for callus induction and regeneration in all explants (Nikam and Shitole 1998). The effect of NaCl and polyethylene glycol on callus growth parameters of two safflower cultivars was determined. Callus was initiated on MS medium supplemented with various concentrations of BAP and NAA also containing different concentrations of NaCl. In these experiments, two safflower cultivars were considered as first factor. The different concentrations of NaCl and polyethylene glycol (PEG) were used as second factor (Kakaei et al. 2013).

      The effects of pyruvic acid, ferulic acid, and cinnamic acid on growth and α-tocopherol and pigment productions in C. tinctorius callus and cell suspension cultures were evaluated. The callus and cell suspension growth and α-tocopherol and pigment contents improved significantly on MS medium containing NAA and BAP during one month of incubation period. Incorporation of pyruvic acid significantly enhanced the production of α-tocopherol and yellow pigment. Similarly, supplementation of pyruvic acid enhanced red pigment in callus culture. The cinnamic acid also significantly improved the production of yellow and red pigments in cell suspension culture. However, precursor feeding suppresses the biomass of cell culture and production of α-tocopherol in cell suspension culture (Başalma et al. 2008; Chavan and Nikam 2015).

      Safflower cell cultures cultured in the presence of different concentrations of cadmium were assayed for growth, Cd accumulations, and antioxidative responses. Cadmium inhibited the growth of calli by nearly 50% with supplementation of different Cd concentrations. Calli accumulated Cd per kg of their dry weight at 100 μM Cd concentration. Cadmium induced oxidative stress, which was indicated by modulating antioxidant levels and antioxidative enzymes. The effect of Cd on glutathione was dose dependent. The glutathione content increased up to a concentration of 75 μM Cd and then reduced. Levels of α-tocopherol showed a significant increase with the increase in concentrations of Cd in the media. Antioxidant enzyme activity increased significantly up to a concentration of Cd. Concentrations of Cd greater than 75 μM resulted in a decline in antioxidant enzyme activity (Namjooyan et al. 2012).

References

      1 Ali, N. and Afrasiab, H. (2014). Effect of TIBA and other plant growth regulators on callogenic response from different explants of safflower (Carthamus tinctorius). Int. J. Agric. Biol. 16: 1112–1116.

      2 Amini, H., Arzani, A., and Bahrami, F. (2013). Seed yield and some physiological traits of safflower as affected by water deficit stress. Int. J. Plant Prod. 7: 597–614.

      3 Asgarpanah, J. and Kazemivash, N. (2013). Phytochemistry, pharmacology and medicinal properties of Carthamus tinctorius L. Chin. J. Integr. Med. 19: 153–159.

      4 Bajji, M., Bertin, P., Lutts, S., and Kinet, J.M. (2004). Evaluation of drought resistance-related traits in durum wheat somaclonal lines selected in vitro. Aust. J. Exp. Agric. 44: 27–35.

      5 Başalma, D., Uranbey, S., Mirici, S., and Kolsarici, Ö. (2008). TDZ × IBA induced shoot regeneration from cotyledonary leaves and in vitro multiplication in safflower (Carthamus tinctorius L). Afr. J. Biotechnol. 7: 960–966.

      6 Chavan,

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