Production of plumbagin from cell cultures of Plumbago rosea L.

Callus and suspension cultures derived from leaf explants of Plumbago rosea were established and plumbagin, a naphthoquinone, was isolated from them and confirmed by ¹H NMR and electron-ionization mass spectroscopy. Maximum content of plumbagin was obtained in the stationary phase of growth (4.3 mg g^–1 dry cell wt). Media pH, phytohormones and carbon sources were optimized for biomass and plumbagin accumulation. Cell aggregates, measuring 500 m in diam, produced 8.2 g dry cell wt l^–1, but larger aggregates (above 500 m) favored plumbagin accumulation with an yield of 4.5 mg g^–1 dry cell wt.     

Rapid clonal propagation of Plumbago zeylanica Linn.

An efficient protocol was developed for in vitro clonal propagation of Plumbago zeylanica Linn. through nodal culture. Multiple shoots were induced from nodal explants of P. zeylanica on Murashige and Skoog's (1962) medium supplemented with 0.5 mg L^–1 to 1.0 mg.L^–1 6-benzyladenine and 3% (w/v) sucrose. Inclusion of IAA (0.01 mg L^–1) in the culture medium improved the frequency of production of multiple shoots. Rooting was readily achieved upon transferring the shoots onto half-strength MS medium supplemented with 0.25 mg L^–1 IBA and 2% (w/v) sucrose. Micropropagated plantlets were hardened in the greenhouse and successfully established in soil.     

Micropropagation ofGmelina arborea

Multiple shoots were obtained from single node explants of matureGmelina arborea Roxb. on MS medium supplemented with 6-benzyladenine (BA). Seven to nine shoots were formed whenin vitro-derived single node explants were subcultured on MS medium supplemented with 1.1 M BA. For root initiation the cut ends of microshoots were pulsed for 5 min with 246 M indole-3-butyric acid and transferred to a plastic cup containing sterile vermiculite. The shoots were covered with polyethylene bag and maintained in a culture room. After hardening, plantlets were transferred to earthen pots containing a mixture of garden soil: compost and have been established in the field.     

In vitro propagation of the medicinal plant Plumbago zeylanica L. Through nodal explants

Summary A protocol for rapid in vitro propagation using nodal explants obtained from 2-yr-old, field-grown medicinal plants of Plumbago zeylanica L. belonging to the family Plumbaginaceae is described. High frequency bud break and fast development of shoots were induced on Murashige and Skoog's basal medium supplemented with 27.2 μM adenine sulfate +2.46 μM indole-3-butyric acid (IBA). Induction of rooting was achieved by transferring the shoots to the same basal medium containing 4.92 μM IBA. Using our protocol from one twig of P. zeylanica (eight responsive nodes per explant shoot) within a period of 5 mo., eight plantlets could be raised. After a hardening period of 4 wk, there was a 90% transplantation success in the field compared to the 60–65% survival of plantlets recorded in the experiments of previous workers. The plantlets derived through in vitro propagation mimic the growth and morphological characteristics of the donor plants.     

Acetylsalicylic acid and ammonium-induced somatic embryogenesis and enhanced plumbagin production in suspension cultures of <Emphasis Type="Italic">Plumbago rosea</Emphasis> L.

Summary Somatic embryogenesis was induced from suspension cultures (derived from leaf callus) of an important medicinal plant, Plumbago rosea L. While acetylsalicylic acid (ASA) alone induced embryogenesis, indole-3-acetic acid (IAA) failed to elicit a similar response. This is the first time that ASA-induced somatic embryogenesis has been reported in cultured cells. Optimal embryogenic response per culture was observed in Murashige and Skoog’s medium containing a combination of ASA (8.32 μM) and IAA (5.06 μM). but 1-naphthaleneacetic acid and indole-3-butyric acid individually did not induce somatic embryogenesis. Increase in the concentration of ammonium enhanced the number of embryos formed per culture. Accumulation of plumbagin, an important naphthoquinone and a medicinal compound, was three times higher in embryogenic compared to non-embryogenic suspensions.     

Micropropagation of Cardiospermum Halicacabum

The in vitro studies with Cardiospermum halicacabum indicated that the different explants, i.e cotyledon, hypocotyl, cotyledonary node, leaf, internode and node had the potential to produce calli on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP) and napthalene acetic acid (NAA). Calli of different explant origin showed variable growth responses on different BAP concentrations. The shoots were favourably formed from the calli of leaf and cotyledon explants. The maximum number of shoots were produced from calli subcultured on MS + BAP (17.8 µM). The roots were initiated on growth regulator free MS medium.     

Plant regeneration from stem nodal segments of <Emphasis Type="Italic">Orthosiphon stamineus</Emphasis> benth., a medicinal plant with diuretic activity

Summary A micropropagation method for Orthosiphon stamineus, using stem nodal segments, has been developed. The highest number of regenerated shoots was obtained on Murashige and Skoog (MS) medium supplemented with 6.7 μM benzyladenine with the formation of an average of 6.1 shoots per explant over a period of 4 wk. The number of shoots increased with longer culture duration on proliferation medium. Multiple shoots which were maintained on the proliferation medium for 6 wk had the highest proliferation rate. Separation of multiple shoots and culturing in larger flasks significantly promoted the growth and formation of plantlets. All the in vitro plantlets survived when transferred to the field and showed no significant morphological differences from the mother plants.     

Micropropagation ofMinthostachys mollis (H.B.K.) grieseb. and essential oil composition of clonally propagated plants

Summary Cultures ofMinthostachys mollis were established from nodal explants obtained under aseptic conditions. Explants were cultured on Murashige and Skoog (MS) half-strength medium containing 6-benzyladenine (BA), and/or naphthaleneacetic acid (NAA). Optimum numbers of healthy shoots were induced on media containing 0.05 μM NAA plus 2.2 μM BA; higher concentrations caused more hyperhydricity and less extension. Rooting was achieved on half-strength MS medium with 0.05 μM NAA. Plantlets were acclimatized and successfully transferred to soil. Essential oil composition of the regenerated plants were determined by gas chromatography/mass spectrometry and little differences were found in the essential oil composition with the plant grown in the wild.     

Physiological comparisons of transformed (crown gall) and nontransformedVinca rosea L. Cells

Summary In vitro growth rates of transformed (crown gall) and nontransformed cultures ofVinca rosea L. were greater at 32°C than at 25°C. The growth of transformed cells was significantly inhibited by kanamycin, neomycin, and chloramphenicol but not by cycloheximide. Nontransformed cells were inhibited by all four antibiotics., The relative growth rates of transformed cultures induced by four different strains, ofAgrobacterium tumefaciens did not correspond to the relative rates of tumor weight increase observed in vivo nor with the relative weights of tumor tissue in, plants 8 weeks after inoculation with the corresponding bacterial strains.     

Micropropagation ofTribulus terrestris L., an important medicinal plant

Direct regeneration of shoots and roots through juvenile expiants has been achieved inTribulus terrestris. Cotyledonary leaves along with epicotyl segment from young seedlings were cultured on MS medium containing various concentrations of auxin with cytokinin and glutamine. A combination of 0.2 mgL⁻¹ NAA, 0.5 mgL⁻¹ BAP and 50 mgL⁻¹ glutamine induced high frequency of shoot and root differentiation in 10 weeks. The callus also could be induced on the above medium from the cut end of radical segments. Morphogenic response such as per cent shoot and root differentiation was recorded at regular intervals.     

Effect of Plumbago zeylanica extract and certain curing agents on multidrug resistant bacteria of clinical origin

Alcoholic extract of Plumbago zeylanica (root) was tested against multidrug-resistant clinical isolates of bacteria (Salmonella paratyphi, Staphylococcus aureus, Escherichia coli, Shigella dysenteriae and a R-plasmid-harbouring standard strain, E.coli x⁺). The extract exhibited strong antibacterial activity against all test bacteria irrespective of their antibiotic resistance behaviour. Phytochemical analysis of crude extract revealed the presence of flavonoids, saponins and naphthoquinone. A comparative evaluation of R-plasmid elimination from E. coli x⁺ (pUK 651) by the plant extract, DNA intercalating dyes (acridine orange and ethidium bromide) and a DNA gyrase antagonizing drug (pefloxacin) were made. All these agents could cure R-plasmid effectively at their respective sub-MIC concentrations. Maximum plasmid curing was observed by pefloxacin (88%), followed by ethidium bromide (36%), acridine orange (14%) and alcoholic extract of P. zeylanica (14%). Curing of plasmid pUK651 from E. coli x⁺ was confirmed by determining the loss of resistance markers in the cured derivative culture. This revised version was published online in November 2006 with corrections to the Cover Date.     

Micropropagation of Sesbania aculeata (Pers.) by adventitious organogenesis

Multiple shoots differentiated from hypocotyl explants of Sesbania aculeata (Pers.) syn S. cannabina (Retz.) Pers., a leguminous woody shrub, when cultured on Murashige and Skoog's basal medium supplemented with auxin (IBA, NAA) or auxin and cytokinin (IBA + BAP, NAA + BAP). Shoot budding occurred directly from the explant as well as from callus. Differentiation of shoot and root occurred in one step in the same concentration of auxin or auxin and cytokinin. Elongation of shoots occurred in the shoot induction medium.     

Micropropagation of adult avocado

A procedure has been developed to successfully micropropagate the IV-8 selection, an adult avocado rootstock. Cultures were initiated from basal shoots obtained after pruning a tree back to ground level. Buds sprouted in Murashige and Skoog solid medium with macroelements at half strength and a 1.3 μM benzyladenine supplement. To induce proliferation, shoots were cultured for 2 weeks in liquid medium in a rollordrum with the Gamborg salt formulation and 1.3 μM benzyladenine, followed by 6 weeks in double phase conditions (solid medium with a layer of liquid medium on the top) using the same salt formulation and two different benzyladenine supplements, 2.8 μM in the solid phase and 0.4 μM in the liquid phase. Ninety percent of the shoots rooted after a 3-day culture in liquid medium in a rollordrum with MS macroelements at 0.3× and 4.9 μM indolebutyric acid, followed by transfer to solid medium in the absence of auxin but with 1 g l⁻¹ activated charcoal. Survival rate during the acclimatization in the greenhouse was 70%. This revised version was published online in June 2006 with corrections to the Cover Date.     

Micropropagation of Cypripedium flavum through multiple shoots of seedlings derived from mature seeds

Cypripedium flavum, known as the rare lady’s slipper orchid, is one of the endemics with a yellow flower in China. Due to its conservation and commercial requirement, establishment of an efficient method for micropropogation is urgently needed. Multiple shoots were obtained by placing seedlings from seeds of C. flavum on Harvais media supplemented with two cytokinins (BAP or KIN) used alone or in addition to different concentration of potato homogenate. The effect of BAP was better than that of KIN on shoot multiplication. The Havais media supplemented with BAP (2.22 μM) and potato homogenate (20 g l⁻¹) was the most effective, providing high shoot multiplication frequencies (95%) associated with a high number of shoots per explant (2.55 shoots/plant). For root formation, high rooting and survival were achieved using 1/2 Harvais media supplemented with 0.6 g l⁻¹activated charcoals. High-level activated charcoal increased the number and the length of roots because the activated charcoal could absorb BAP in the media. This study demonstrated that C. flavum could be micropropagated by using multiple shoots of seedlings derived from mature seeds.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Micropropagation of Eclipta alba and Eupatorium adenophorum using a single-step nodal cutting technique

Protocols for the micropropagation of two traditional medicinal plants Eclipta alba (L.) and Eupatorium adenophorum (L.) from nodal segments were developed. Proliferated microshoots of Eclipta alba and Eupatorium adenophorum were obtained through axillary branching by culturing nodal segments in modified MS medium and half strength of MS, respectively, with minimal strength of nutritional support. Simultaneous rooting could also be induced in the same medium. Regenerated rooted plantlets were successfully acclimatized in soil where they grew normally without showing any morphological variation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Keratinolytic activity of Kocuria rosea

A strain of Kocuria rosea able to secrete keratin-hydrolysing proteinases (keratinases) in submerged batch cultures with finely milled feathers as carbon and nitrogen sources was studied. The highest production of keratinases was obtained when feathers were used as the only fermentation substrate (17 U/mg). Considerably lower activity was present in cultures containing glucose and others nutrient supplements. The optimum temperature and pH for keratinolytic activity was 40 °C and 10, respectively. Gelatin-sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) electrophoresis analysis showed that Kocuria rosea grown on feathers secreted at least two alkaline extracellular proteases with apparent molecular weights of 90.2 and >200 kDa, respectively. These proteolytic activities appear sequentially during microbial growth. Keratinolytic activity was strongly inhibited by phenylmethanesulphonyl fluoride (PMSF), chymostatin and crystalline soybean trypsin inhibitor, indicating the presence of serine proteases. Proteolytic enzymes derived from the biodegradation of feathers by this microorganism could be a useful biotechnological tool in the leather, food and cosmetic industries.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.     

Polarity of nuclear and organellar DNA during megasporogenesis and megagametogenesis in Plumbago zeylanica

Summary Megasporogenesis and megagametogenesis of Plumbago zeylanica were studied using isolated megasporocytes, megaspores, and embryo sacs labeled with Hoechst 33258 for nuclear and organellar (presumably plastid) DNA. Megasporogenesis conforms to the tetrasporic Plumbago type, producing a coenomegaspore with four megaspore nuclei. Organeller DNA is polarized in the micropylar end of the coenomegaspore and embryo sac, reflecting the site of egg cell formation. The three remaining nuclei are somewhat displaced to the chalazal pole, producing a variable number of accessory cells and a 4N secondary central cell nucleus. Ultimately, the mature embryo sac consists of two to five cells including an egg cell, a central cell, zero to two lateral cells, and zero to one antipodal cell depending on the degeneration of the lateral or chalazal nuclei during megagametogenesis.     

A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica

Plumbago zeylanica is a unique model for studying flowering plant gametogenesis, heterospermy, and preferential fertilization, yet understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. We found three key factors for enhancing successful regeneration: (1) tissue source of explants, (2) combination and concentration of growth regulators, and (3) culture conditions. The highest frequency of shoot regeneration was achieved using hypocotyl segments cultured on MS basal medium supplemented with BA 2.0 mg/l, NAA 0.75 mg/l, adenine 50 mg/l and 10% (v/v) coconut milk under subdued light at 25±2°C; under these conditions, each hypocotyl segment produced over 30 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Regenerated shoots rooted easily on half-strength basal MS medium and were successfully established in the greenhouse. Using this tissue culture protocol, reporter gene GUS under the constitutive CaMV 35S promoter was introduced into P. zeylanica cells of petiole, cotyledon and hypocotyl with A. tumefaciens strains AGL1 and LBA4404. Transient expression was observed in all recipient tissues. Stable transgenic calli originating from petiole were obtained.