Effect of the NO donor sodium nitroprusside (SNP), the ethylene inhibitor cobalt chloride (CoCl2) and the antioxidant vitamin E α-tocopherol on in vitro shoot proliferation of Sideritis raeseri Boiss. & Heldr. subsp raeseri

In the present study, the effects of the NO donor sodium nitroprusside (SNP) and the ethylene inhibitor CoCl2 combined with 2.2 mu M 6-benzyladenine (BA), and the antioxidant vitamin E alpha-tocopherol in combination with 2.2 mu M zeatin (ZT) and 0.2 mu M alpha-naphthaleneacetic acid (NAA) on in vitro shoot proliferation of Sideritis raeseri Boiss. & Heldr. subsp. raeseri, one of the seven endemic mountain tea species of the Greek flora were investigated. The basal nutrient culture medium used in all experiments was the MS (Murashige, Skoog, Physiol Plant 15:473-497, 1962). Taking simultaneously into consideration all shoot proliferation parameters evaluated, the explants performance was optimum and significantly better among others when treated with 2.2 mu M BA + 10 mu M SNP or 2.2 mu M BA + 2.5 mu M CoCl2, or 2.2 mu M ZT + 0.2 mu M NAA + 11.6 mu M alpha-tocopherol. In particular, 10 mu M SNP gave 4.6 +/- 0.6 shoots/explant and 100% shoot multiplication percentage while 20 mu M SNP exhibited the greatest shoot length (17.5 +/- 1.8 mm). However, SNP (10-40 mu M) caused hyperhydricity symptoms to the 30-100% of explants. CoCl2 applied at 2.5 mu M led to the production of 5.5 +/- 0.7 shoots/explant, 11.8 +/- 1.1 mm shoot length and 100% shoot multiplication percentage and concurrently decreased the percentage of vitrified explants from 33.3 to 20%. 11.6 mu M alpha-tocopherol gave 6.7 +/- 0.8 shoots/explant, 14.5 +/- 0.7 mm shoot length, 100% shoot multiplication percentage, diminishing vitrification percentage from 41.6 to 8.3% (33.3% decrease) and completely inhibiting the appearance of necrotic explants, indicating its antioxidant properties as a vitamin. Shoot-tip explants responded better and exhibited better shoot proliferation results when cultured in MS medium with 2.5 mu M CoCl2 for 5 weeks or 11.6 mu M alpha-tocopherol for 9 weeks rather than with SNP for 4 weeks. In this study, three new different protocols were established for the in vitro shoot proliferation stage of S. raeseri.