induced by plant hormone, jasmonates. To improve the production of the pharmaceuticals, the jasmonate biosynthesis-related gene allene oxide cyclase from C. acuminata was transferred back into C. acuminata by Agrobacterium-mediated genetic transformation and overexpressed. The camptothecin content of transgenic callus was higher than that of nontransgenic callus. However, camptothecin content in both transgenic and nontransgenic calli significantly decreased after further extrinsic MeJA's induction, whereas the content of camptothecin in transgenic callus was still higher than in nontransgenic one. All the results indicate that endogenic jasmonate's accumulation may be promoted after allene oxide cyclase gene was transformed into C. acuminata and overexpressed (Pi et al. 2012).
Hairy roots derived from the wild-type strains of Agrobacterium rhizogenes have also been developed in C. acuminata; the yields of the camptothecin in the two species were of approximately 1 mg/g dry weight (DW). The highest camptothecin content of the transgenic callus reached 3.9310 mg/g DW in this present research, which was higher than the one reported. The highest growth rate of callus was observed on MS medium with NAA, kinetin, and sucrose. All tissues and organs developed in vitro contain camptothecin and 10-hydroxycamptothecin. The presence of 10-hydroxycamptothecin in shoots and callus of C. acuminata Decne is reported for the first time (Wiedenfeld et al. 1997). The effects of I−,
Camptothecin, a well-known monoterpenoid indole alkaloid originally identified in the extracts of the Chinese tree C. acuminata, exhibits antitumor activity due to its ability to kill cancer cells via topoisomerase I poisoning. Other plant species have since been shown to produce camptothecin and related compounds (Yamazaki et al. 2003). The complementary DNAs for strictosidine synthase, tryptophan decarboxylase (TDC), and cytochrome P450 reductase were cloned from Ophiorrhiza pumila and evaluated for involvement in production of camptothecin in this species. RNA interference-mediated knockdown of gene expression indicated that the production of camptothecin, strictosidine, and camptothecin-related alkaloids was suppressed in a TDC expression-dependent manner in RNA interference hairy roots (Liu et al. 1998; Lorence and Nessler 2004; Yamazaki et al. 2013).
The young flower buds had the highest alkaloid concentrations (camptothecin and 10-hydroxycamptothecin). Callus showed lower concentrations, but it should also be considered as a potential source of these pharmaceuticals (Lorence et al. 2004). The growth rate of C. acuminata cells in culture did not correlate with the contents of camptothecin and 10-hydroxycamptothecin (Lopez-Mayer et al. 1997; Zhang et al. 2007; Pi et al. 2010).
Camptothecin production in regenerated plants was compared with its production in calli and the original seedlings. Dark incubation and seed coat removal led to a higher germination rate and a higher survival rate after germination. The best shoot induction medium was found to be Gamborg's medium + benzyl adenine. The leaf petiole calli had a higher shoot regeneration rate and a higher shoot number than did leaf disc calli on the best shoot induction medium. It took four to six weeks to regenerate shoots after transfer into shoot induction media. Camptothecin concentration in the regenerated plants was significantly higher than that in the calli and similar to that in the original seedlings (Li and Liu 2005).
Concentrations of the DNA topoisomerase I inhibitor camptothecin were determined in different tissues of C. acuminata. Despite the presence of the toxic compound camptothecin, nine endophytic fungi were isolated from healthy C. acuminata plants and were tested for their camptothecin sensitivity. Even at a concentration of 100 μg/ml, the inhibition was moderate (Liu and Reinscheid 2004). Similarly, (Wiedenfeld et al. 1997) in callus cultures of C. acuminata and in shoot cultures has reported a low amount of camptothecin (Roja 2008).
The effect of plant growth regulators on the accumulation of the alkaloid camptothecin and its analogue 10-hydroxycamptothecin in the tender leaves of C. acuminata saplings was studied. In screening experiments for plant growth regulators, thiourea, triacontanol, and ascorbic acid had no positive effects on the accumulation of the alkaloids. However, treatments with 40 chlormequat chloride, choline chloride, paclobutrazol, and daminozide (B9) induced camptothecin and 10-hydroxycamptothecin accumulation in both preharvest and postharvest stages. Various concentrations of growth hormones were sprayed on tender leaves of C. acuminata saplings at preharvest and postharvest stages (Liu and Adams 1998). Treatment of chlormequat chloride dramatically enhanced 10-hydroxycamptothecin production by 308% in preharvest and by 100% in postharvest. In the preharvest treatment, spraying the leaves with choline chloride resulted in 94% increase of camptothecin, and spraying with growth hormones reached 167% increase of 10-hydroxycamptothecin; treatments with choline chloride resulted in maximum increase of camptothecin and 10-hydroxycamptothecin in postharvest. Treatments with daminozide had the highest impact on camptothecin and 10-hydroxycamptothecin accumulation. The optimal concentration of growth hormones for obtaining the highest levels of camptothecin and 10-hydroxycamptothecin was determined. In most cases, the preharvest treatment was better than the postharvest one. These preliminary results suggest that the application of plant growth regulators may be a useful and feasible method to increase camptothecin and 10-hydroxycamptothecin levels in C. acuminata (Zeng et al. 2012).
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