The organogenic competence of different explants of Rhaponticum carthamoides was investigated on MS agar medium supplemented with BA, IBA or NAA at concentrations of 0.2 and 0.5 mg L -1 . Adventitious shoot formation was obtained through direct organogenesis using leaves of in vitro cultures as explants and through indirect organogenesis when seedling explants (hypocotyl, cotyledon and root) were used for regenerative callus initiation. The shoots were rooted on half-strength MS medium ( MS) without auxin or containing IBA (0.2-2.0 mg L -1 ). The plantlets regenerated through direct and indirect organogenesis were transferred into pots and grown in the greenhouse for 3 months. Significant differences in morphology, accumulation of chlorogenic acid and 20-hydroxyecdysone (20-HE) as well as in genetic profile were observed between these plants. UHPLC analysis showed that the highest level of chlorogenic acid (12 mg g -1 DW) was found in leaves of plants developed directly from explants, whereas leaves of plants developed via callus tissue accumulated the highest amount of 20-HE (7.4 mg g -1 DW). Its level exceeded that detected in leaves of 3-month-old plants obtained from seeds (2.4 mg g -1 DW). Genetic variations of R. carthamoides regenerated plants were evaluated by flow cytometry and RAPD and ISSR methods. Flow cytometry confirmed similar ploidy level in the mother plant and plants regenerated through direct and indirect organogenesis. Genetic similarity values calculated on the basis of RAPD and ISSR data among regenerated in vitro plants to the mother plant were ranged from 0.765 to 0.941 and 0.647 to 0.947, respectively.
The effects of thidiazuron (TDZ) pretreatment of shoot tips on Harpagophytum procumbens shoot proliferation and successive stages of micropropagation, i.e. rooting of regenerated shoots and acclimatization of plantlets to ex vitro conditions, were described in the present study. The best response in terms of shoot proliferation (about seven shoots/explant) and shoot length (3.2 ± 0.4 cm) was obtained when explants pretreated with 25 lmol L -1 TDZ for 6 h were cultured on Schenk and Hildebrandt medium containing indole-3-acetic acid (IAA) (0.57 lmol L -1 ) and 6-benzylaminopurine (BAP) (8 lmol L -1 ). Under these conditions, a 330 % increase in shoot multiplication over TDZ non-pretreatment culture was achieved and TDZ pretreatment shoots were longer compared to those in control culture (2.6 ± 0.3 cm). The TDZ pretreatment did not affect the percentage of rooted shoots, length of roots and number of roots formed per shoot. The rooted plantlets were transplanted from in vitro to pots with soil and grown during 1 year in the greenhouse. The hardening process was difficult and time-consuming. We found that the plants developed from the TDZ pretreated culture were superior to plants from non-pretreated culture in terms of survival rate and morphological features, such as shoot length, leaf size, flowering and earlier root tuberisation. Random amplified polymorphic DNA and inter-simple sequence repeat analyses of pretreatment with TDZ plants showed genetic similarity to non-pretreatment plants. We conclude that applying the strategy of initial explant pretreatment with TDZ may be valuable for the improvement in H. procumbens in vitro propagation.
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