Biotechnological Advances in Pharmacognosy and In Vitro Manipulation of Pterocarpus marsupium Roxb.

Trees are vital resources for economic, environmental, and industrial growth, supporting human life directly or indirectly through a wide variety of therapeutic compounds, commodities, and ecological services. Pterocarpus marsupium Roxb. (Fabaceae) is one of the most valuable multipurpose forest trees in India and Sri Lanka, as it is cultivated for quality wood as well as pharmaceutically bioactive compounds, especially from the stem bark and heartwood. However, propagation of the tree in natural conditions is difficult due to the low percentage of seed germination coupled with overexploitation of this species for its excellent multipurpose properties. This overexploitation has ultimately led to the inclusion of P. marsupium on the list of endangered plant species. However, recent developments in plant biotechnology may offer a solution to the overuse of such valuable species if such advances are accompanied by technology transfer in the developing world. Specifically, techniques in micropropagation, genetic manipulation, DNA barcoding, drug extraction, delivery, and targeting as well as standardization, are of substantial concern. To date, there are no comprehensive and detailed reviews of P. marsupium in terms of biotechnological research developments, specifically pharmacognosy, pharmacology, tissue culture, authentication of genuine species, and basic gene transfer studies. Thus, the present review attempts to present a comprehensive overview of the biotechnological studies centered on this species and some of the recent novel approaches for its genetic improvement.

Auxin-cytokinin synergism in vitro for producing genetically stable plants of Ruta graveolens using shoot tip meristems.

An efficient micropropagation protocol was developed for Ruta graveolens Linn. using shoot tip meristems derived from a 4-month-old field grown plant. In vitro shoot regeneration and proliferation was accomplished on Murashige and Skoogs (MS) semi-solid medium in addition to different doses of cytokinins viz.6- benzyl adenine (BA), Kinetin (Kn) or 2-isopetynyl adenine (2iP), singly or in combination with auxins viz. indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) or α-naphthalene acetic acid (NAA). Highest regeneration frequency (27.6%) was obtained on (MS) medium composed of BA (10 µM) with maximum number (9.4) of shoots and 4.3 cm shoot length after 4 weeks of incubation. Among various combinations tried best regeneration frequency (71%) of multiple shoot formation with highest number (12.6) of shoots per shoot tip explants were achieved in MS medium augmented with a combination BA (10.0 µM) and NAA (2.5 µM) after 4 weeks of incubation. The optimum frequency (97%) of rhizogenesis was achieved on half-strength MS medium having 0.5 µM IBA after 4 weeks of incubation. Tissue culture raised plantlets with 5-7 fully opened leaves with healthy root system were successfully acclimatized off in Soilrite™ with 80% survival rate followed by transportation to normal soil under natural light. Genetic stability among in vitro raised progeny was evaluated by ISSR and RAPD markers. The entire banding pattern revealed from in vitro regenerated plants was monomorphic to the donor. The present protocol provides an alternative option for commercial propagation and fruitful setting up of genetically uniform progeny for sustainable utilization and germplasm preservation.

Embling Production in Althaea officinalis L., Through Somatic Embryogenesis and Their Appraisal via Histological and Scanning Electron Microscopical Studies.

In vitro propagation of a medicinally important plant, Althaea officinalis, has been achieved through somatic embryogenesis. Somatic embryos (globular to torpedo-shaped embryos) were induced on Murashige and Skoog's (MS) medium augmented with various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D, 5.0, 10.0, 15.0, 20.0, and 25.0) alone or combined with N6-benzylaminopurine (BA, 0.1, 0.5, 1.0, 1.5, and 2.0 µM). These were directly formed from the cut ends and subsequently spread on the whole surface of internodal explants. For embryo maturation, torpedo embryos were transferred on a medium containing different levels of BA (0.1, 0.5, or 1.0 µM) and abscisic acid (ABA) (0.5, 1.0, or 1.5 µM) or α-naphthalene acetic acid (NAA) (0.1, 0.5 or 1.0 µM). Among the different concentrations tested, 0.5 µM BA along with 1.0 µM ABA was found most effective, on which a highest yield (58.0%) with an optimum number (35.0) of mature embryos (cotyledonary stage) was observed after 2 weeks of transfer. Germination of cotyledonary embryos into plantlets with 68% were observed on ½ MS medium. Histological and scanning electron microscopical (SEM) studies proved that the regenerated structures were somatic embryos and not shoot primordia. Plants grew vigorously when transferred to a greenhouse.

Retraction Note to: influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types.

[This retracts the article DOI: 10.1007/s12298-012-0136-4.].

In Vitro Propagation and Conservation of Withania somnifera (Dunal) L.

Plant tissue culture offers several techniques for rapid clonal propagation, germplasm conservation, regeneration of genetically manipulated superior clones, production of phyto-constituents, and ex vitro conservation of valuable phytodiversity. An improved and efficient micropropagation protocol for Withania somnifera (L.), a drug-producing medicinal plant, using juvenile explants (nodal explants) has been developed. Highest multiplication and subsequent elongation of shoots is observed on MS medium containing BA and NAA. The regenerated microshoots roots best on ½ MS medium containing NAA, established in earthen pots containing garden soil and are maintained in the greenhouse with 95 % survival rate. Genetic uniformity of micropropagated plants is confirmed by PCR-based DNA fingerprinting techniques, viz., RAPD and ISSR. No variation is observed in DNA fingerprinting patterns among the micropropagated plants, which are similar to that of the donor plant illustrating their genetic uniformity.

Interactive Effects of Growth Regulators, Carbon Sources, pH on Plant Regeneration and Assessment of Genetic Fidelity Using Single Primer Amplification Reaction (SPARS) Techniques in Withania somnifera L.

An improved and methodical in vitro shoot morphogenic approach through axillary bud multiplication was established in a drug yielding plant, Withania somnifera L. Effects of plant growth regulators [6-benzyladenine (BA), kinetin (Kin), 2-isopentenyladenine (2iP), and thidiazuron (TDZ)] either singly or in combination with α-napthalene acetic acid (NAA), indole-3-butyric acid (IBA), and indole-3-acetic acid (IAA) in Murashige and Skoog (MS) medium were tested. The highest regeneration frequency (90 %) with optimum number of shoots (32 ± 0.00)/explant were obtained on MS medium fortified with 2.5 µM 6-benzyladenine (BA) and 0.5 µM NAA and 30 g/l sucrose at pH 5.8. Among the tried TDZ concentrations, 0.5 µM resulted in maximum number of shoots (20.4 ± 0.40)/explant after 4 weeks of exposure. The proliferating shoot cultures established by repeated subculturing of the mother explants on the hormone-free medium produced the highest shoot number (29.4 ± 0.40) with shoot length (6.80 ± 0.12 cm)/explant at fourth subculture passage, which a decline in shoot proliferation was recorded. Different concentrations of NAA were tested for ex vitro rooting of microshoots. The maximum percentage of rooting 100 % with maximum roots (18.3 ± 0.1) was achieved in soilrite when basal portion of the microshoots were treated with 200 µM (NAA) for 15 min per shoot. The plantlets went through hardening phase in a growth chamber, prior to ex vitro transfer. The PCR-based single primer amplification reaction (SPAR) methods which include random amplified polymorphic DNA (RAPD) and direct amplification of minisatellite DNA (DAMD) markers has been used for assessment of genetic stability of micropropagated plantlets. No variation was observed in DNA fingerprinting patterns among the micropropagated and the donor plants illustrating their genetic uniformity.

An efficient in vitro process for cyclic clonal production of shoots from adult tree of Cassia alata L. and evaluation of genetic stability using DNA-based markers.

An efficient, cyclic, two-step protocol for clonal in vitro regeneration system of an antiallergenic plant, Cassia alata, has been successfully developed. Nodal explants from a 5-year-old tree were cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5, and 10.0 µM) of thidiazuron (TDZ). TDZ (5.0 µM) was found to be optimal for the formation of maximum shoot induction. Shoot proliferation and elongation increased when the regenerated shoots were subcultured on hormone-free MS medium after 4 weeks of exposure to TDZ. Nodal explants from in vitro regenerated microshoots to developed shoots, thus making the process recurrent. In 6 months duration, owing to the recurring nature of the protocol, large number of shoots could be produced from a single nodal explant from an adult tree. Shoots rooted best on MS supplemented medium with 0.5 µM IBA. Regenerated plantlets were acclimatized and successfully transplanted to the garden soil, where they grew well without any morphological and genetic variations. To confirm the uniformity, the genetic fidelity of in vitro raised C. alata clones was also assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. The present regeneration process not only favored the clonal multiplication but also expressed the regeneration capability of in vitro regenerated microshoots and can be subjugated for catering enough raw materials to various pharma industries by continuous cyclic shoot production.

Rapid in vitro propagation system through shoot tip cultures of Vitex trifolia L.-an important multipurpose plant of the Pacific traditional Medicine.

A rapid and efficient plant propagation system through shoot tip explants was established in Vitex trifolia L., a medicinally important plant belonging to the family Verbenaceae. Multiple shoots were induced directly on Murashige and Skoog (MS) medium consisting of different cytokinins, 6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP), BA at an optimal concentration of 5.0 µM was most effective in inducing multiple shoots where 90 % explants responded with an average shoot number (4.4±0.1) and shoot length (2.0±0.1 cm) after 6 weeks of culture. Inclusion of NAA in the culture medium along with the optimum concentration of BA promoted a higher rate of shoot multiplication and length of the shoot, where 19.2±0.3 well-grown healthy shoots with an average shoot length of 4.4±0.1 cm were obtained on completion of 12 weeks culture period. Ex vitro rooting was achieved best directly in soilrite when basal portion of the shoots were treated with 500 µM indole-3-butyric acid for 15 min which was the most effective in inducing roots, as 95 % of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered with 92 % survival rate. The results of this study provide the first report on in vitro plant regeneration of Vitex trifolia L. using shoot tip explants.

Effect of adenine sulphate interaction on growth and development of shoot regeneration and inhibition of shoot tip necrosis under in vitro condition in adult Syzygium cumini L.--a multipurpose tree.

An efficient method for cloning Syzygium cumini (above 40 years old) through mature nodal segments has been successfully developed and that could be exploited for large-scale production of this valuable multipurpose tree. Nodal segments from mature tree were taken as explants and cultured on MS basal medium with different cytokinins (BA, Kin, AdS). The application of BA proved to be the best responsive cytokinin for the induction of shoot buds and shoots, but the proliferated shoots exhibited slower and stunted growth accompanied with abscission of leaves and shoot tip necrosis (STN). The problem of leaf abscission and STN was considerably reduced by the application of an adjuvant, adenine sulphate (AdS) in the optimal medium which led to the production of a maximum of 14 shoots. Further improvement in shoot bud regeneration and improved growth pattern of the regenerating tissue was obtained on the media comprised of MS + BA (10 µM) + GA3 (2.5 µM). A total number of 15 shoots with mean shoot length of 5.9 cm was obtained. The healthy elongated shoots were then rooted on MS basal augmented with NAA (5 µM). The plantlets obtained were healthy and were successfully acclimatized and transferred under field condition with 70 % survival rate.

Relative examination of antioxidative enzymatic activities in plantlets of Cardiospermum halicacabum L. differentiated from hypocotyls in in vivo and ex vitro environment.

A plant regeneration protocol was devised for Cardiospermum halicacabum by means of aseptically extracted 7 days old hypocotyls forming adventitious shoots on Murashige and Skoog (MS) medium harmonized with 0.7 µM thidiazuron (TDZ) producing a maximum of 18.20 ± 0.98 number of shoots in 94% cultures following 4 weeks. Subsequent subculturing for five passages, on a medium without plant growth regulators, tempted the highest shoot number (40.00 ± 1.15) with an average shoot length of 6.53 ± 0.49 cm after the fourth subculture. Histological sections confirmed the formation of multiple buds from hypocotyl explants. The expression of antioxidant enzymes like superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase was found to be higher in acclimatized plants than in the in vitro cultured ones suggesting the involvement of these enzymes in shoot differentiation and in growth under external environment partly due to their ability to cope up with oxidative stress.

Role of TDZ in the quick regeneration of multiple shoots from nodal explant of Vitex trifolia L.--an important medicinal plant.

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0 µM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0 µM BA and 0.5 µM NAA. The highest shoot regeneration frequency (90 %) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2 cm) was recorded on MS medium fortified with 5.0 µM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 µM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.

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.

Assessment of genetic fidelity in Rauvolfia serpentina plantlets grown from synthetic (encapsulated) seeds following in vitro storage at 4 °C.

An efficient method was developed for plant regeneration and establishment from alginate encapsulated synthetic seeds of Rauvolfia serpentina. Synthetic seeds were produced using in vitro proliferated microshoots upon complexation of 3% sodium alginate prepared in Llyod and McCown woody plant medium (WPM) and 100 mM calcium chloride. Re-growth ability of encapsulated nodal segments was evaluated after storage at 4 °C for 0, 1, 2, 4, 6 and 8 weeks and compared with non-encapsulated buds. Effects of different media viz; Murashige and Skoog medium; Lloyd and McCown woody Plant medium, Gamborg's B5 medium and Schenk and Hildebrandt medium was also investigated for conversion into plantlets. The maximum frequency of conversion into plantlets from encapsulated nodal segments stored at 4 °C for 4 weeks was achieved on woody plant medium supplement with 5.0 µM BA and 1.0 µM NAA. Rooting in plantlets was achieved in half-strength Murashige and Skoog liquid medium containing 0.5 µM indole-3-acetic acid (IAA) on filter paper bridges. Plantlets obtained from stored synseeds were hardened, established successfully ex vitro and were morphologically similar to each other as well as their mother plant. The genetic fidelity of Rauvolfia clones raised from synthetic seeds following four weeks of storage at 4 °C were assessed by using random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. All the RAPD and ISSR profiles from generated plantlets were monomorphic and comparable to the mother plant, which confirms the genetic stability among the clones. This synseed protocol could be useful for establishing a particular system for conservation, short-term storage and production of genetically identical and stable plants before it is released for commercial purposes.

Influencing micropropagation in Clitoria ternatea L. through the manipulation of TDZ levels and use of different explant types.

A comparative performance of two explants types (CN and Nodal) for their efficiency to induce multiple shoot regeneration in Clitoria ternatea has been carried out. Thidiazuron (TDZ) in different concentrations (0.05-2.5 µM) was used as a supplement to the Murashige and Skoog's (MS) basal media. Explant type apart, two factors viz. concentration and exposure duration to TDZ played an important role in affecting multiple shoot regeneration. Cotyledonary node explants produced the best results at 0.1 µM TDZ, while in nodal explants the highest rate of shoot formation was achieved on MS medium supplemented with 1.0 µM TDZ. In both the explants, shoot multiplication increased when the regenerated shoots were subcultured on hormone free MS medium after 4 weeks of exposure to TDZ. Among the two, cotyledonary node explants produced considerably higher number of shoots at a comparatively lower concentration of TDZ than nodal explants. The regenerated shoots rooted best on MS medium containing 1.0 µM indole-3-butyric acid (IBA) and were successfully established in pots containing garden soil with 88 % survival rate. All the regenerated plants showed normal morphology and growth characteristics.

Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal.

An efficient, rapid and improved in vitro plant regeneration protocol has been established for Withania somnifera L. using shoot tip and nodal explants, excised from 15 days old aseptic seedlings. A range of cytokinins were investigated for multiple shoot regeneration. Of the three cytokinins, 6-benzyladenine (BA), Kinetin (Kin) and 2-isopentenyl adenine (2-iP) evaluated as supplement to Murashige and Skoog (MS) medium, BA at an optimal concentration of 2.5 µM was most effective in proliferating apical and axillary buds. The highest regeneration frequency (95 %) and number of shoots (36.1 ± 0.33) were obtained on MS medium fortified with BA (2.5 µM) and NAA (0.5 µM) from nodal segments. High frequency of rooting (100 %) was obtained in in vitro raised shoots when transferred to half-strength MS medium supplemented with NAA (0.5 µM). Histological sections revealed that additional shoot bud primordia were differentiated within the explants just underneath the suberized cells which appeared to be arrested in their development. The presence of additional bud primordia within the explants is thereby helpful to maximize the potential of this system. The regenerated plantlets with well developed shoots and roots were hardened successfully, established in earthen pots containing garden soil and maintained in greenhouse with 95 % survival rate.

Enhanced in vitro regeneration and change in photosynthetic pigments, biomass and proline content in Withania somnifera L. (Dunal) induced by copper and zinc ions.

In the present study the effect of inorganic nutrients (CuSO4 & ZnSO4) on morphogenic and biochemical responses from nodal explants in Withania somnifera L. was investigated. Incorporation of either Copper sulphate (25-200 µM) or Zinc sulphate (50-500 µM) in the optimized Murashige and Skoog (MS) medium highly influenced the shoot bud formation and subsequent elongation, which induced maximum percentage (95%) regeneration, number (61.7 ± 0.25) of shoots with shoot length (5.46 ± 0.16 cm) on CuSO4 (100 µM) and maximum percentage regeneration (100%), number of shoots (66.1 ± 0.96) with shoot length (6.24 ± 0.21 cm) on ZnSO4 (300 µM) after 12 weeks of culture. Healthy growing in vitro microshoots rooted efficiently on ½ MS medium supplemented with NAA (0.5 µM), which induced (16.2 ± 0.12) roots with root length (3.30 ± 0.12 cm) after 4 weeks. Pigment content increased with increasing concentration of Cu and Zn and the maximum Chl. a (0.47), (0.41); Chl. b (0.52), (0.42); total Chl. (0.99), (0.83) and Carotenoid (0.16), (0.16) mg/g FW contents in regenerants were found on CuSO4 (100 µM) and ZnSO4 (300 µM), respectively. Maximum proline content (0.17), (0.16) µg/g FW was observed on high concentrations of CuSO4 (200 µM) and ZnSO4 (500 µM) respectively, in the basal medium. Regenerated plantlets were acclimatized successfully in soilrite with a survival rate of 95%. No morphological variations were detected among the micropropagated plants when compared with seedling raised plants of the same age.