Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant.

Rauwolfia tetraphylla L. is an important medicinal plant species which is well known for its pharmaceutically important alkaloids. In the present study, we are reporting about its conservation by in vitro clonal multiplication through the standardized protocol of indirect regeneration by using leaf and stem based callus and assessment of genetic fidelity of acclimated plantlets by start codon targeted (SCoT), inter simple sequence repeats (ISSR), and randomly amplified polymorphic DNA (RAPD) marker based analysis. Initially friable callus was induced in maximum amounts (378.7, 323.8, and 412.8 in mg) from leaf, root, and stem explants on Murashige and Skoog (MS) media supplemented with 5.0 mg/L, 3.0 mg/L of 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.0 mg/L of naphthalene acetic acid (NAA), respectively. Shoot regeneration with the maximum number of shoot buds (25 and 20) was obtained from leaf and stem calluses on MS media supplemented with TDZ (0.25 mg/L) + BAP (2 mg/L). The regenerated shoots were rooted successfully with maximum rooting percentage of 98.0 on full strength MS media amended with IAA (1.0 mg/L) and IBA (1.0 mg/L). The regenerated plantlets were hardened using 2:1 ratio of sterile garden soil and sand, followed by acclimatization in field conditions with 86% of survival. SCoT, ISSR, and RAPD primers based polymerase chain reaction (PCR) analysis was carried out to check possible genetic variations in micro propagated plants in comparison with mother plant. Among the ten SCoT (S), ISSR (R), and RAPD (OPA) primers used, S2, R10, and OPA3 has given good amplification with scorable DNA bands. The results revealed that the regenerated plants did not have any polymorphism with mother plant. Hence, the in vitro regenerated R. tetraphylla plantlets were confirmed as true-to-type.

An efficient and reproducible method for in vitro clonal multiplication of Rauvolfia tetraphylla L. and evaluation of genetic stability using DNA-based markers.

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 µM BA, 2.5 µM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 µM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

The influence of different hormone concentration and combination on callus induction and regeneration of Rauwolfia serpentina L. Benth.

The influence of media composition on callus induction and subsequent regeneration of Rauwolfia serpentina L. Benth has been studied. High frequency (96.43%) callus induction was obtained when nodal segments from in vitro raised shoots were cultured on MS medium supplemented with 0.5 mg L(-1) BA and 2.0 mg L(-1) NAA. The callus differentiated into adventitious shoots when it was subcultured on MS medium supplemented with 2.0 mg L(-1) BA with 0.2 mg L(-1) NAA. Regenerated shoots were best rooted on half-strength MS medium with 1.0 mg L(-1) each of IBA and IAA.

Cryopreservation of in vitro grown nodal segments of Rauvolfia serpentina by PVS2 vitrification.

This paper describes the cryopreservation by PVS2 vitrification of Rauvolfia serpentina (L.) Benth ex kurz, an important tropical medicinal plant. The effects of type and size of explants, sucrose preculture (duration and concentration) and vitrification treatment were tested. Preliminary experiments with PVS1, 2 and 3 produced shoot growth only for PVS2. When optimizing the PVS2 vitrification of nodal segments, those of 0.31 - 0.39 cm in size were better than other nodal sizes and or apices. Sucrose preculture had a positive role in survival and subsequent regrowth of the cryopreserved explants. Seven days on 0.5 M sucrose solution significantly improved the viability of nodal segments. PVS2 incubation for 45 minutes combined with a 7-day preculture gave the optimum result of 66 percent. Plantlets derived after cryopreservation resumed growth and regenerated normally.

Increase of free cysteine and citric acid in plant cells exposed to cobalt ions.

Cobalt complexation was investigated in a suspension cell culture of the cobalt hyperaccumulator Crotalaria cobalticola. C. cobalticola cells were more tolerant towards cobalt ions than the suspension cells of the non-accumulators Rauvolfia serpentina and Silene cucubalus. While the concentration of various compounds increased in cells of C. cobalticola challenged with cobalt ions, phytochelatin biosynthesis was not induced. Instead, the exposure to cobalt ions resulted in the increase of citrate and cysteine in cells. Size exclusion chromatography demonstrated the co-elution of cobalt and cysteine in C. cobalticola cell extracts. A significant increase in cysteine was observed also in cells of R. serpentina and S. cucubalus when they were exposed to cobalt ions. These results suggest that free cysteine is involved in cobalt ion complexation in plant cells.

[Effect of phytohormones on the protein-synthesizing ability of rauwolfia serpentina benth. tissue culture]. / Vliianie fitogormonov na beloksinteziruiushchuiu sposobnost' kul'tury tkani Rauwolfia serpentina Benth.

The accumulation of intracellular protein by the callus culture of Rauwolfia serpentina Benth. was studied in a standard or phytohormone-containing medium. Changes in the concentration of total protein in cells induced by indolylacetic and naphthylacetic acids were shown to be associated with the effects of these phytohormones on the biosynthesis and degradation of intracellular protein.

In vitro propagation of Rauvolfia serpentina through tissue culture.

Multiple shoots were induced from nodal segments and shoot apices of Rauvolfia serpentina and MS medium containing 1.0 mg/l BA and 0.1 mg/l NAA was found to give the best shoot proliferation rate. Callus formed at cut bases of the explants which produced shoots when subcultured on media containing low concentration of BA (0.5 or 0.1 mg/l) and NAA (0.1 mg/l). The in vitro proliferated shoots were rooted and later transferred to the soil.