An efficient protocol for in vitro regeneration from the nodal explants of Withania coagulans (Stocks) Dunal: a valuable medicinal herb

<p>In order to develop a protocol for the effective micropropagation of the important medicinal plant Withania coagulans (Stocks) Dunal, the effects of different concentrations and combinations of growth regulators on the nodal explants in two independent experiments were investigated. For shooting, a MS medium fortified with different concentrations and combinations of IBA (0.01, 0.1 and 0.5 mg l-1), BA (0.5, 1 and 2 mg l-1), Kin (0.5 and 1 mg l-1), PG (0.5 mg l-1) and GA (0.5 mg l-1) was used and the highest shooting response, shoot number and shoot length were obtained in the MS + IBA (0.01 mg l-1) + BA (0.5 mg l-1) + PG (0.5 mg l-1) + GA (0.5 mg l-1) treatment. In the second experiment, the effect of MS supplemented with different combinations and concentrations of IBA (0.1, 0.5, 1 and 2 mg l-1), NAA (0.1 and 1 mg l-1) and PG (1 mg l-1) on rooting of the nodal explants was investigated, which showed that the highest rooting response (%) was observed in the MS fortified with NAA (0.1 mg l-1), NAA (1 mg l-1), NAA (0.1 mg l-1) + PG (1 mg l-1), and NAA (1 mg l-1) + PG (1 mg l-1) treatments, as well as the highest number of roots at NAA (0.1 mg l-1) and the highest root length at IBA (1 mg l-1). Our findings highlight a complete micropropagation method for W. coagulans from the nodal explant that can make a significant contribution to the development of W. coagulans material for medical applications.</p>

Regeneration of Hemidesmus indicus (L.) R. Br. using in vitro nodes: an alternative method for efficient multiplication of shoots

In vivo nodes of Hemidesmus indicus (L.) R. Br. induced healthy multiple shoots with branching in our earlier studies and thus in the present study, potency of in vitro nodes to regenerate shoots was evaluated. In vitro nodes were excised from eight-week-old shoots and placed in Murashige and Skoog’s (MS) medium fortified with sucrose (3%) and different concentrations of 6-benzyladenine (BA) and kinetin (Kn). After eight weeks, optimum of 5.42 ± 0.36 shoots with 100% response were regenerated in medium supplemented with BA (10 µM) and Kn (5 µM). These healthy shoots were placed in full, half and quarter strengths of liquid MS medium fortified with sucrose (1%) and α-naphthaleneacetic acid (NAA, 1-25 µM) for rooting. Among all the strengths of MS medium, full strength MS medium having 8 µM NAA formed maximum of 3.42 ± 0.55 roots (91.67% response) within four weeks. The protocol is in continuation with earlier study and it was confirmed that a single in vivo nodal explant can regenerate around 385 healthy elongated shoots within 4 months, which will help in mass-propagation of the species.

Surface sterilization method for reducing contamination of Clinacanthus nutans nodal explants intended for in-vitro culture

Surface sterilization is a vital step in preparation of healthy and viable explants in tissue culture. Most surface contaminants can be eliminated by surface sterilization with a suitable sterilizing agent. The study aimed to present an effective disinfection method for Clinacanthus nutans shoot regeneration using nodal segments. A total of four different sterilization approaches were conducted by treating nodal explants with various concentrations of sterilizing agent. Sterilizing agents used were Rhizophora apiculata Pyroligneous acid (PA), sodium hypochlorite (Clorox) thiophanate-methyl (fungicide), and Mercuric chloride (HgCl2). Nodal explant then was cultured on plant growth regulator-free Murashige and Skoog (MS) basal medium. This study sterilizing agents revealed that PA showed strong bactericidal activity. However, it led to a high number of fungal contaminations. The pyroligneous acid did not exhibit a strong potential as a disinfectant for C. nutans nodal explant. Overall, HgCl2 exhibits the best reduction in fungal contamination and gives a significant result with thiophanate-methyl fungicide. Surface sterilization with mercuric chloride (0.2%) for 1 hour was the optimum concentration and duration, which resulted in the highest percentage of nodal explant survival and viability. All viable nodal segments developed into shoots. It had been concluded that the best surface sterilization agent was HgCl2.

Citrus TISSUE CULTURE WITH TWO DIFFERENT APPROACHES

Citrus is an important genius for economy and human health but a susceptible genius against biotic and abiotic stresses, so it needs to improved programs. Different basal media (MS, ½MA, ?MS and DKW) and different kind and concentrations of plant growth regulators i.e. BA, KIN, 2ip, ZE and TDZ (0 – 2 mg/l) and NAA, IAA and IBA (0 – 2 mg/l) added with 30 g/l sucrose, 3 g/l active charcoal and 7.5 g/l bacteriological agar] were used for organogenesis include shooting and rooting, also callusing from nodal explant of Citrus latifolia. MS medium supplemented with 1 mg/l BA, and 0.01 mg/l NAA is the best media for multiple shoot induction on nodal explants and elongation of them. Other cytokinins had not significant effects on shoot induction and multiplication. Using of 0.01 mg/l IBA instead of 0.01 mg/l NAA on medium with 1 mg/l BA, led to multiple shoot induction on nodal explant indirectly. Rooting was induced on DKW medium plus 1.5 mg/l NAA in the best way compared another media. Both direct and indirect organogenesis (multiple shoot induction) were carried out on media with very similar contents. So we can use very simple and practical methods tissue culture for different improved programs in Citrus genius genius.

Study on in vitro propagation of Giao co lam (Gynostemma pentaphyllium Thunb.)

Giao co lam (Gynostemma pentaphyllium Thunb.) is a traditional medicine plant and endangered species in Vietnam. The research was carried out to establish the plant propagation for the purpose of conserving and exploiting this endangered medicinal herb. The young leaf and nodes of Giao co lam in vitro were used as explants in the study to evaluate the factors influencing the multiplication. Young leaf explants were excised and cultured in MS medium with TDZ from 0.1 to 1 mg/L. After 10 weeks of culture, new shoots came out from their explants and the MS medium containing TDZ 0,7 mg/L gave the highest shoots (12.89 shoots/explant) with the average percentage of 74.67%. When nodal explants were cultured on MS supplemented with BA at a concentration of 0.3 to 1.5 mg/L and IBA 0.5 mg/L. After 6 weeks of culture, explants on MS medium supplemented with BA 1 mg/L and IBA 0,5 mg/L gave the highest shoots (7.39 shoots/explant) and their average percentage was 83.33%. In comparison to the nodal explant medium in combination with BA (0.5 to 3 mg/L) and NAA 0.2 mg/L for 4 weeks of culture, the best rapid shoot multiplication score was 3.67 times with MS + BA 1.5 mg/L + NAA 0.2 mg/L as compared to MS + BA 1.0 mg/L + IBA 0.5 mg/L. Suitable medium for rooting was MS + 0.5 mg/L IBA with root shoots at 97.33%, average roots at 5.29 roots/shoot after 4 weeks. On organic substrat, 70% coconut fiber and 30% composted cow manure gave the highest survival rate of 91.33%. The plants grew and developed well during the nursery stage.