<Emphasis Type="Italic">In vitro</Emphasis> propagation of the genus <Emphasis Type="Italic">Clivia</Emphasis>

We have developed a protocol for the in vitro propagation of the genus Clivia. Shoots were regenerated when fragments of the peduncle-pedicel junction (PP junction) from young inflorescences were used as explants. The optimal media for PP junction were Murashige and Skoog (MS)-based medium containing 10 M of 6-benzyladenine (BA) and 10 M of 2,4-dichlorophenoxyacetic acid (2,4-D) or MS supplemented with 5 M BA, 10 M -naphthaleneacetic acid (NAA), 250 mg l^-1 glutamine and 500 mg l^–1 casein hydrolysate and their usage depended on the breeding lines. Multiplication from initiations and in vitro seedlings was the best when the explants were cut longitudinally through the meristem and placed on MS plus 44 M BA. Plantlets were transferred on to hormone -free MS medium with charcoal for rooting.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of Persian poppy (<Emphasis Type="Italic">Papaver bracteatum</Emphasis>)

Persian poppy (Papaver bracteatum Lindl.) is an important commercial source of medicinal opiates and related compounds. In this research, calli were induced from seeds, roots, cotyledons and hypocotyls of P. bracteatum at a high efficiency. The optimized callus induction media consisted of the Murashige and Skoog (MS) basic media supplemented with 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 0.1 mg/L kinetin and 15 mg/L ascorbic acid. The concentrations of 2,4-D and ascorbic acid were found critical to callus induction and proliferation. Subsequent subcultures resulted in excellent callus proliferation. Ascorbic acid at concentration 15 mg/L increased the callus proliferation significantly. Maximum callus growth was achieved when the explants were incubated at 25°C. MS salts at full strength were found inhibitory for callus induction, while ľ MS salts were found to favor callus induction. Shoot regeneration of calli in vitro was achieved on ľ MS medium containing 0.5 mg/L benzylamine purine and 1.0 mg/L naphthalene acetic acid. Analysis of alkaloid extracts from Persian poppy tissues by high-performance liquid chromatography showed that thebaine accumulated in the tissues of plants. The thebaine alkaloid profile of the Persian poppy is a well-defined model to evaluate the potential for metabolic engineering of thebaine production in P. bracteatum.     

Morphological evaluation and antioxidant activity of <Emphasis Type="Italic">in vitro</Emphasis>- and <Emphasis Type="Italic">in vivo</Emphasis>-derived <Emphasis Type="Italic">Echinacea purpurea</Emphasis> plants

An effective in vitro protocol for rapid clonal propagation of Echinacea purpurea (L.) Moench through tissue culture was described. The in vitro propagation procedure consisted of four stages: 1) an initial stage - obtaining seedlings on Murashige and Skoog (MS) basal medium with 0.1 mg L⁻¹ 6-benzylaminopurine, 0.1 mg L⁻¹ α-naphthalene acetic acid and 0.2 mg L⁻¹ gibberellic acid; 2) a propagation stage — shoot formation on MS medium supplemented with 1 mg L⁻¹ 6-benzylaminopurine alone resulted in 9.8 shoots per explant and in combination with 0.1 mg L⁻¹ α-naphthalene acetic acid resulted in 16.2 shoots per explant; 3) rooting stage — shoot rooting on half strength MS medium with 0.1 mg L⁻¹ indole-3-butyric acid resulted in 90% rooted microplants; 4) ex vitro acclimatization of plants. The mix of peat and perlite was the most suitable planting substrate for hardening and ensured high survival frequency of propagated plants. Significant higher levels were observed regarding water-soluble and lipid-soluble antioxidant capacities (expressed as equivalents of ascorbate and α-tocopherol) and total pnenols content in extracts of Echinaceae flowers derived from in vitro propagated plants and adapted to field conditions in comparison with traditionally cultivated plants.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of endangered <Emphasis Type="Italic"> Rhododendron ponticum</Emphasis> L. subsp. <Emphasis Type="Italic">baeticum</Emphasis> (Boissier &; Reuter) Handel-Mazzetti

In vitro propagation of Rhododendron ponticum L. subsp. baeticum, an endangered species present in limited and vulnerable populations as a Tertiary relict in the southern Iberian Peninsula, was attained. Several cytokinin:IAA ratios and a range of zeatin concentrations were evaluated for their effect on shoot multiplication from apical shoots and nodal segments. The type of cytokinin and the origin of the explant were the most important factors affecting shoot multiplication. The highest shoot multiplication rate was obtained from single-nodal explants on medium supplemented with zeatin. Increasing zeatin concentration promotes shoot multiplication independently of explant type, although this effect tends to decrease with higher zeatin concentration. Shoot growth was higher in apical shoots and it was not stimulated by the presence of auxin. A number of experiments were conducted to identify suitable procedures for rooting of in vitro produced shoots. The best results in terms of in vitro rooting were obtained with Andersons modified medium with macrosalts reduced to one-half, regardless of the auxin or its concentration in the medium. Although rooting frequency rose to 97% by basal immersion of shoots in auxin concentrated solution followed by in vitro culture on an auxin-free medium, the survival of the plants after 6 months of acclimatization was poor (50%). Best results (100% rooting and survival) were observed for ex vitro rooting. The micropropagated plants from this study were successfully reintroduced into their natural habitat (87% of survival after 8 months).     

Overexpression of yeast <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase <Emphasis Type="Italic">metK</Emphasis> in <Emphasis Type="Italic">Streptomyces actuosus</Emphasis> leads to increased production of nosiheptide

S-Adenosylmethionine (SAM) is synthesized via the metabolic reaction involving adenosine triphosphate and l-methionine that is catalyzed by the enzyme S-adenosyl-l-methionine synthetase (SAM-s) and encoded by the gene metK. In the present study, metK with the absence of introns from Saccharomyces cerevisiae was introduced into Streptomyces actuosus, a nosiheptide (Nsh) producer. Intracellular SAM levels were determined by high-pressure liquid chromatography. Through optimizing the nutrient content of the medium, it was shown that increased SAM production induced by the overexpression of SAM-s leads to an increase in the intracellular cysteine pool and overproduction of Nsh in S. actuosus. This investigation shows that increased SAM promotes the elevated production of the non-ribosomal thiopeptide Nsh in Streptomyces sp.     

Approaches on vegetative propagation of difficult-to-root <Emphasis Type="Italic">Salix caprea</Emphasis>

In the framework of a willow rust research project, it was necessary to include vegetatively propagated plant material of selected sallow trees (Salix caprea L.) into biotests for identification of pathotypes. Since it was not possible to root sufficient clonal plants by conventional cutting propagation, the applicability of tissue culture methods was tested. From 10 selected donor trees of Salix caprea newly sprouted shoots were harvested and transferred to nutrient media after surface disinfection. The cultures were grown at 20--22 °C, illuminated with warm-white fluorescent tubes. The majority of shoot tips and nodal segments died during the first month, but only with nursery-grown plants this was caused by bacteria contamination. Two clones could be established easily on hormone-free medium. Five clones could be initiated only after repeated subcultures on various media variants. Three clones failed completely. Different basic media compositions were tested and Woody Plant Medium, supplemented with 0.1% activated charcoal, proved to be best for most of the sallow clones. Well developed rooted plantlets were used in vitro for microcutting propagation. The resulting plants were transferred to soil and could be included in the rust screening program after acclimatising. The applicability of micropropagation for selected Salix caprea donor trees was strongly depending on the genotype. But the comparison of results from microcuttings with conventional cutting propagation showed that these methods were successful for different clones each.     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation from young and mature explants of thyme (<Emphasis Type="Italic">Thymus vulgaris</Emphasis> and <Emphasis Type="Italic">T. longicaulis</Emphasis>) resulting in genetically stable shoots

An efficient in vitro propagation protocol, applicable both to young and mature explants of two Thymus spp., results in genetically stable plantlets. In vitro-grown shoot tips of Thymus vulgaris L. were exposed to cytokinins (6-benzyladenine, kinetin, and thidiazuron) alone or in combination with auxins, gibberellic acid (GA₃) and/or silver nitrate in order to optimize in vitro shoot proliferation. Optimum shoot proliferation (97% regeneration rate, with 8.6 shoots produced per explant) was obtained when semi-solid Murashige and Skoog (MS) medium was supplemented with 1 mg L⁻¹ kinetin and 0.3 mg L⁻¹ GA₃. Rooting of the shoots was easily obtained on semi-solid MS medium that was either hormone-free or supplemented with auxins. However, the best root apparatus (92.5% rooting rate, with 19 adventitious roots per shoot) developed on MS medium supplemented with 0.05 mg L⁻¹ 2,4-dichlorophenoxyacetic acid. Genetic stability was confirmed in the in vitro-germinated mother plant as well as the shoots that underwent two, four, six, eight, or ten cycles of in vitro subculturing by random amplified polymorphic DNA (RAPD) analysis. When applied to the micropropagation of mature shoot tips of T. longicaulis C. Presl subsp. longicaulis var. subisophyllus (Borbás) Jalas, the optimized in vitro propagation protocol resulted in a 97.5% shoot regeneration rate, with five shoots formed per explant, and 100% rooting. Rooted plantlets of both species were transferred to 250-mL plastic pots and successfully acclimatized by gradually reducing the relative humidity.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of the resurrection plant <Emphasis Type="Italic">Haberlea rhodopensis</Emphasis>

The small group of resurrection plants is a unique model which could help us in further understanding of abiotic stress tolerance. The most frequently used approach for investigations on gene functions in plant systems is genetic transformation. In this respect, the establishment of in vitro systems for regeneration and micro propagation is necessary. On the other hand, in vitro cultures of such rare plants could preserve their natural populations. Here, we present our procedure for in vitro regeneration and propagation of Haberlea rhodopensis – a resurrection plant species, endemic for the Balkan region.     

<Emphasis Type="Italic">Acmella radicans</Emphasis> var. <Emphasis Type="Italic">radicans: In vitro</Emphasis> culture establishment and alkamide content

Summary Acmella radicans var. radicans propagation was established in vitro. This plant belongs to the Asteraceae from which some species are known for their insecticide, fungicide and antibacterial activity. The complete Murashige and Skoog (MS) medium was the best in assisting seed germination. In order to obtain shoots in vitro, a complete MS medium and half-strength MS medium were assayed with explants from leaves, nodes, and internodes. The best medium for shoot production was the half-strength MS medium with no addition of plant growth regulators, and the highest shoot propagation was from single-node explants. Regeneration of roots on shoot explants in the medium was obtained without the addition of growth regulators. Of the plantlets that were acclimated, 90% of them were obtained from rooted shoots with completely expanded leaves. The alkamide content was evaluated for each tissue and the higher concentration was observed in flower heads. The main alkamides present in the leaves and the flower heads were N-(2-phenylethyl)-2Z,4E-octadienamide and 3-phenyl-N-(2-phenylethyl)-2-propenamide. This study describes the methodology for the establishment and propagation of Acmella radicans in vitro and the evaluation of different tissue alkamide contents in vitro and in the field.     

Cloning and expression of <Emphasis Type="Italic">mel</Emphasis> gene from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Previous work from our laboratory has shown that most of Bacillus thuringiensis strains possess the ability to produce melanin in the presence of l-tyrosine at elevated temperatures (42 °C). Furthermore, it was shown that the melanin produced by B. thuringiensis was synthesized by the action of tyrosinase, which catalyzed the conversion of l-tyrosine, via l-DOPA, to melanin. In this study, the tyrosinase-encoding gene (mel) from B. thuringiensis 4D11 was cloned using PCR techniques and expressed in Escherichia coli DH5 . A DNA fragment with 1179 bp which contained the intact mel gene in the recombinant plasmid pGEM1179 imparted the ability to synthesize melanin to the E. coli recipient strain. The nucleotide sequence of this DNA fragment revealed an open reading frame of 744 bp, encoding a protein of 248 amino acids. The novel mel gene from B.thuringiensis expressed in E. coli DH5 conferred UV protection on the recipient strain.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation and cryostorage of <Emphasis Type="Italic">Syzygium francissi (Myrtaceae)</Emphasis> by the encapsulation-dehydration method

Summary An efficient procedure for the in vitro propagation and cryogenic conservation of Syzygium francissi was developed. The maximum number of shoots per explant was obtained on a Murashige and Skoog (MS) medium supplemented with 4.5 μM benzyladenine and 0.5 μM indole-3-butyric acid (IBA). The in vitro-propagated shoots produced roots when transferred to MS medium containing IBA, indold-3-acetic acid, or naphthaleneacetic acid at various concentrations. Rooted microshoots were transferred to a coco-peat, perlite, and vermiculite (1∶1∶1) mixture, and hardened off under greenhouse conditions. Ninety-five percent of rooted shoots successfully acclimatized in the greenhouse. Shoot tips excised from in vitro-grown plants were successfully cryostoraged at −196°C by the encapsulation-dehydration method. A preculture of formed beads on MS medium containing 0.75 M sucrose for 1 d, followed by 6 h dehydration (20% moisture content) led to the highest survival rate after cryostorage for 1h. This method is a promising technique for in vitro propagation and cryopreservation of shoot tips from in vitro-grown plantlets of S. francissi germplasm.     

<Emphasis Type="Italic">In vitro</Emphasis> culture of <Emphasis Type="Italic">Chrysanthemum</Emphasis> plantlets using light-emitting diodes

Effects of illumination spectrum on the morphogenesis of chrysanthemum plantlets (Chrysanthemum morifolium Ramat. ‘Ellen’) grown in vitro were studied using an illumination system consisting of four groups of light-emitting diodes (LEDs) in the following spectral regions: blue (450nm), red (640nm), red (660nm), and far-red (735nm). Taking into account all differences in shoot height, root length, and fresh and dry weight (FW and DW, respectively), observed while changing the total photon flux density (PFD), the optimal total PFD for growth of chrysanthemum plantlets in vitro was estimated. For 16 h photoperiod and typical fractions of the spectral components (14%, 50%, 28%, and 8%, respectively), the optimal total PFD was found to be 40 μmol m⁻² s⁻¹. Our study shows that the blue component in the illumination spectrum inhibits the plantlet extension and formation of roots and simultaneously increases the DW to FW ratio and content of photosynthetic pigments. We demonstrate photomorphogenetic effects in the blue region and its interaction with the fractional PFD of the far-red spectral component. Under constant fractional PFD of the blue component, the root number, length of roots and stems, and fresh weight of the plantlets have a correlated nonmonotonous dependence on the fractional PFD of the far-red component.     

Double deletion of <Emphasis Type="Italic">dtsR1</Emphasis> and <Emphasis Type="Italic">pyc</Emphasis> induce efficient <Emphasis Type="SmallCaps">l</Emphasis>-glutamate overproduction in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Corynebacterium glutamicum strains are used for the fermentative production of l-glutamate. Five C. glutamicum deletion mutants were isolated by two rounds of selection for homologous recombination and identified by Southern blot analysis. The growth, glucose consumption and glutamate production of the mutants were analyzed and compared with the wild-type ATCC 13032 strain. Double disruption of dtsR1 (encoding a subunit of acetyl-CoA carboxylase complex) and pyc (encoding pyruvate carboxylase) caused efficient overproduction of l-glutamate in C. glutamicum; production was much higher than that of the wild-type strain and ΔdtsR1 strain under glutamate-inducing conditions. In the absence of any inducing conditions, the amount of glutamate produced by the double-deletion strain ΔdtsR1Δpyc was more than that of the mutant ΔdtsR1. The activity of phosphoenolpyruvate carboxylase (PEPC) was found to be higher in the ΔdtsR1Δpyc strain than in the ΔdtsR1 strain and the wild-type strain. Therefore, PEPC appears to be an important anaplerotic enzyme for glutamate synthesis in ΔdtsR1 derivatives. Moreover, this conclusion was confirmed by overexpression of ppc and pyc in the two double-deletion strains (ΔdtsR1Δppc and ΔdtsR1Δpyc), respectively. Based on the data generated in this investigation, we suggest a new method that will improve glutamate production strains and provide a better understanding of the interaction(s) between the anaplerotic pathway and fatty acid synthesis.     

Genotyping of somatic hybrids between <Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb. and <Emphasis Type="Italic">Triticum aestivum</Emphasis> L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Zingiber petiolatum</Emphasis> (Holttum)

Summary We describe an in vitro propagation protocol for Zingiber petiolatum (Holttum), I. Theilade, a rare species from the southern part of Thailand. Fruits were surface-sterilized and seeds germinated on Murashige and Skoog medium (MS) medium supplemented with 3% sucrose. Three-month-old seedlings were used as initial plant material for in vitro propagation. Terminal buds of the plants were inoculated on MS medium containing 6-benzylaminopurine (BA; 2.2–35.5 μM) alone or in combination with 1-naphthaleneacetic acid (0.5 μM). Eight weeks after inoculation, the cultures were transferred to MS medium without plant growth regulators for 4wk. The cultures transferred from MS medium with 17.8 μM BA revealed the highest shoot induction rate of 6.1±0.7 shoots per explant. Rooting was spontaneously achieved in MS medium without plant growth regulators. Rooted plants were successfully transplanted to soil.     

HPLC and GC analyses of in vitro-grown leaves of the cancer bush <Emphasis Type="Italic">Lessertia</Emphasis> (<Emphasis Type="Italic">Sutherlandia</Emphasis>) <Emphasis Type="Italic">frutescens</Emphasis> L. reveal higher yields of bioactive compounds

Lessertia frutescens L., commonly known as cancer-bush, is a medicinally reputed plant species indigenous to southern Africa. Field leaf extracts of this species are known to exhibit many curative properties. However, little is known about the bioactive compounds that are present in in vitro leaf extracts and seed extracts. The objective of this study was to verify the presence of and quantify l-canavanine, gamma amino butyric acid (GABA), arginine and d-pinitol in the seeds, field leaves and in vitro leaves of L. frutescens using gas and liquid chromatography. Methanolic extracts of in vitro leaves, field leaves and seeds were used. MRM chromatograms were recorded for l-canavanine and arginine using tandem mass spectrometry. GC chromatograms were recorded for GABA and d-pinitol using gas chromatography. d-Pinitol was found to be most abundant and was 14.75 and 18.17 mg/g in in vitro and field leaf extracts respectively, followed by GABA (7.29 and 3.48 mg/g), arginine (7.08 and 0.35 mg/g) and l-canavanine (0.55 and 0.08 mg/g). In the seed extracts, GABA content was found to be the highest (1.69 mg/g) followed by l-canavanine (0.37 mg/g), then d-pinitol (0.25 mg/g), and arginine (0.02 mg/g). In vitro leaves had higher quantities of all compounds, except for d-pinitol. This study therefore highlights the potential of bulking in vitro leaves for the extraction of the medicinal compounds, l-canavanine and GABA.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of North American ginseng (<Emphasis Type="Italic">Panax quinquefolius</Emphasis> L.)

North American ginseng (NAG) (Panax quinquefolius L.) is a medicinally important plant with multiple uses in the natural health product industry. As seed propagation is time-consuming because of the slow growth cycle of the plant, in vitro propagation using a bioreactor system was evaluated as an effective approach to accelerate plant production. An efficient method was developed to multiply nodal explants of NAG using liquid-culture medium and a simple temporary immersion culture vessel. The effects of plant growth regulators, phenolics, and chemical additives (activated charcoal, melatonin, polyvinylpolypyrrolidone, and ascorbic acid) were evaluated on in vitro-grown NAG plants. The highest number (12) of shoots per single node was induced in half-strength Schenk and Hildebrandt basal medium containing 2.5 mg/l kinetin, in which 81% of the cultured nodes responded. In a culture medium with 0.5 mg/l α-naphthalene acetic acid (NAA), roots were induced in 78% of the explants compared to 50% with a medium containing indole-3-acetic acid. All of the resulting plants appeared phenotypically normal, and 93% of the rooted plants were established in the greenhouse. Phenolic production increased significantly (P < 0.05) over a 4-wk culture period with a negative impact on growth and proliferation. Activated charcoal (AC; 50 mg/l) significantly reduced total phenolic content and was the most effective treatment for increasing shoot proliferation. Shoot production increased as the phenolic content of the cultures decreased. The most effective treatment for NAG development from cultured nodal explants in the bioreactor was 2.5 mg/l kinetin, 0.5 mg/l NAA, and 50 mg/l AC in liquid culture medium. This protocol may be useful in providing NAG tissues or plants for a range of ginseng-based natural health products.     

Efficient <Emphasis Type="Italic">in vitro</Emphasis> bulblet regeneration from immature embryos of endangered <Emphasis Type="Italic">Sternbergia fischeriana</Emphasis>

Sternbergia fischeriana is an endangered geophyte and therefore in vitro micropropagation of this plant will have great importance for germplasm conservation and commercial production. Bulb scale and immature embryo explants of S. fischeriana were cultured on different nutrient media supplemented with various concentrations of plant growth regulators. Immature embryos produced higher number of bulblets than bulb scales. Large numbers of bulblets were regenerated (over 80 bulblets/explants) from immature embryos on Murashige and Skoog (MS) medium supplemented with 4 mg l^–1 6-benzylaminopurine (BA) and 0.25 mg l^–1 -naphthaleneacetic (NAA) or 2 mg l^–12,4-dichlorophenoxyacetic acid (2,4-D) after 14 months of culture initiation. Regenerated bulblets were kept at 5 °C for 5 weeks and then transplanted to a potting mixture.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of medicinal plant <Emphasis Type="Italic">Centella asiatica</Emphasis>

This paper describes multiple shoot regeneration from leaf and nodal segments of a medicinally important herb Centella asiatica L. on Murashige and Skoog’s (MS) medium supplemented with a range of growth regulators. The highest number of multiple shoots was observed on MS augmented with 3.0 mg dm⁻³ N⁶-benzylaminopurine (BAP) and 0.05 mg dm⁻³ α-naphthaleneacetic acid (NAA). Leaf explant showed maximum percentage of cultures regenerating shoots (81.6 %), with the highest shoot number (8.3 shoots per explant) and the shoot length (2.1 cm) whereas, nodal explant showed less number of shoots with callus formation at the base cut end. Successive shoot cultures were established by repeatedly sub-culturing the original explant on a fresh medium. Rooting of in vitro raised shoots was best induced on half strength MS supplemented with 0.5 mg dm⁻³ indole-3-butyric acid (IBA) with highest percentage of shoot regenerating roots (76.8 %) with 3–4 roots per shoot. Plantlets were acclimated in Vermi-compost and eventually established in soil. Contents of chlorophyll, total sugars, reducing sugars and proteins were estimated in leaf tissue from both in vivo and in vitro raised plants. Chlorophyll content was higher in in vivo plants, whereas other three components were higher in in vitro plants.