Genome size analysis of field grown and tissue culture regenerated Rauvolfia serpentina (L) by flow cytometry: Histology and scanning electron microscopic study for in vitro morphogenesis
2019
Zafar, Nadia | Mujib, A. | Ali, Muzamil | Tonk, Dipti | Gulzar, Basit | Malik, Moien | Sayeed, Rukaya | Mamgain, Jyoti
An efficient genetically stable regeneration protocol has been established in Rauvolfia serpentina (L) Benth ex. Kurz, an important medicinal plant, used for the treatment of various cardiovascular diseases. Here, in this present study, plants were regenerated through organogenesis (direct and indirect) and embryogenesis pathways. This is the first ever somatic embryo formation report of R. serpentina in liquid Murashige and Skoog (MS) medium from isolated root segments. The genetic fidelity of the regenerated plants was also analysed by flow cytometry. The 2C DNA content of the regenerated plants raised through organogenesis and somatic embryogenesis was measured along with field grown natural plants, which served as donor plant. The estimated 2C DNA levels are 2.08, 1.76 and 1.88 pg in direct, indirect and somatic embryo regenerated plants respectively while in field grown plant it was noted to be 1.94 pg in R. serpentina. This is perhaps the first ever 2C DNA estimation study in tissue culture raised plants along with natural population of Rauvolfia, an immensely important medicinal plant, present in India. We developed improved callus mediated shoot regeneration (indirect) protocol from leaf callus. Highest shoot regeneration percentage (66.10%) with 4.0 shoots / callus mass was noted on 2.0 mg l⁻¹ BAP + 0.5 mg l⁻¹ NAA added MS medium. The shoot tips and nodal explants on 2.5 mg l⁻¹ BAP amended MS medium induced direct shoot buds, with highest shoot regeneration frequency (81.45%) from shoot tips. On 1.0 mg l⁻¹NAA added MS medium maximum numbers of roots (8.87) were noted, indicating the effectiveness of NAA over IBA in rooting. Excised roots were cultured in agitated liquid MS medium and in 1.0 mg l⁻¹ NAA added conditions somatic embryos were formed. The mature somatic embryos germinated on 1.5 mg l⁻¹ BAP + 0.25 mg l⁻¹ GA₃ supplemented MS medium. The histology and scanning electron microscopic (SEM) evidences have been presented to describe the in vitro morphogenesis process. The well-rooted plantlets were acclimatized and transferred to soil successfully (79%). The flow cytometric analysis showed no significant variation in nuclear DNA contents, hence was observed to be genetically stable with their genome size similar to field grown Rauvolfia plants.
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