Expression of a KDEL-tagged dengue virus protein in cell suspension cultures of <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> and <Emphasis Type="Italic">Morinda citrifolia</Emphasis>

Hypericum perforatum L. (St. John’s wort) produces a number of phytochemicals having medicinal, anti-microbial, anti-viral and anti-oxidative properties. Plant extracts are generally used for treatment of mild to medium cases of depression. Plant regeneration can be achieved in this species by in vitro culture of a variety of explants. However, there are no reports of regeneration from petal explants. In this report plant regeneration from petal explants of St. John’s wort was evaluated. Petals of various ages were cultured on agarized Murashige and Skoog 1962 (MS) medium supplemented with auxin and cytokinin (kinetin), maintained in the dark and callus and shoot regeneration determined after 28 days. At an auxin to cytokinin ratio of 10:1, callus and shoot formation were induced by all levels of indole-3-acetic acid (IAA), indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), while 2,4-dichlorophenoxyacetic acid (2,4-D) induced only callus formation. The optimum level of auxin for shoot regeneration was 1.0 and 0.1 mg/l kinetin, where the regeneration frequency was 100 percent for all three auxins. The highest number of shoots per explant (57.4 and 53.4) was obtained with IAA and IBA, respectively. In the absence of auxin, kinetin levels of 0.1 and 0.25 mg/l induce callus and shoot formation at low frequency but not at lower levels. Callus and shoot formation did not occur in the absence of growth regulators. Petal-derived shoots were successfully rooted on half-strength MS medium without a requirement for exogenous auxin and flowering plants were established under greenhouse conditions. From these results it can be concluded that auxin type is a critical factor for plant regeneration from petal explants of Hypericum perforatum and there is no absolute requirement for high levels of cytokinin.     

Plant regeneration from callus and protoplast cultures of Lotus pedunculatus Cav.

Plant regeneration from leaf- and cotyledon-derived calli and from protoplast-derived tissue has been obtained in Lotus pedunculatus. Callus induction was achieved with 2,4-D and plant regeneration required the following two media sequences: bud formation was stimulated by IAA and BA and shoot growth by kinetin. Root formation occurred in the presence of IAA. Cotyledon protoplasts showed a low plating efficiency and plant regeneration was achieved via an intervening callus phase.Abbreviations BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2iP N^6--2-isopentenyl-adenine - NAA -naphthaleneacetic acid     

Regeneration of niger (Guizotia abyssinica Cass.) CV Sahyadri from seedling explants

Root, hypocotyl and cotyledonary explants of niger (Guizotia abyssinica Cass) CV. Sahyadri were aseptically cultured on Murashige and Skoog's basal medium (MS) containing BAP and kinetin. Multiple shoot regeneration was induced from hypocotyl and cotyledonary explants while root explants produced only callus on MS medium supplemented with BAP. BAP (1 mg l^-1) was optimum for shoot regeneration. Regenerated shoots were transferred to MS medium without auxins, with auxins and with increasing concentrations of sucrose for rooting. Complete plantlets were obtained in all cases; however, 0.5 mg l^-1 NAA was the best for induction of roots. Ninety-seven per cent of the plantlets survived and completed their life cycle when transferred to natural conditions.Abbreviations BAP 6-benzylamino purine - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid     

Mass propagation of <Emphasis Type="Italic">Plectranthus bourneae</Emphasis> Gamble through indirect organogenesis from leaf and internode explants

The present study describes the plant propagation via indirect organogenesis from in vitro derived leaf and internode explants of Plectranthus bourneae, an endemic plant to south India. Leaf and internodal explants successfully callused on Murashige and Skoog medium (MS) supplemented with different concentrations of auxins [2,4-D (2,4-dichlorophenoxyacetic acid), NAA (α-naphthalene acetic acid), IAA (indole-3 acetic acid), IBA (indole-3-butyric acid) and PIC (Picloram); 0.1–2.0 mg/l] in combination with BA (6-benzyladenine) (0.5 mg/l). Maximum callus induction (98 %) was achieved from leaf explant followed by internodal explant (89 %) at 1.0 mg/l NAA, 0.5 mg/l BA. Leaf derived callus showed better shoot regeneration (29.71 shoots) on MS medium containing 1.0 mg/l KN (kinetin), 0.7 mg/l NAA, and 50 mg/l CH (casein hydrolysate) followed by internodal callus (19.71). A maximum of 19.14 roots/shoot was observed at 1.0 mg/l IBA. The rooted plantlets were successfully hardened and transferred to greenhouse condition with 80 % survival. This system could be utilized for large-scale multiplication of P. bourneae by tissue culture.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae)

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or -naphthaleneacetic acid (0.5–10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5–5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or -napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5–2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0–5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with -naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80–90%).Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MAP 6-methylaminopurine - MS Murashige and Skoog (1962) medium - NAA -naphthaleneacetic acid - PVP polyvinyl pyrrolidone     

Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas

Techniques for the regeneration of Jatropha curcas L. from various explants have been developed. Regeneration from hypocotyl, petiole and leaf explants was evaluated on a range of concentrations of zeatin, kinetin and N⁶ benzyladenine (BA) either singly or in combination with indole-3-butyric acid (IBA). Higher regeneration from hypocotyl and petiole explants was obtained on BA with IBA than on zeatin- or kinetin-supplemented media. Leaf discs from the third expanding leaf exhibited higher regeneration potential than those from the fourth leaf. Independent of the explant type, direct adventitious shoot bud induction was recorded highest on MS medium with 2.22 M BA and 4.9 M IBA. Although the same BA concentration but with reduced IBA concentration (0.49 M) proved effective in callus mediated regeneration from hypocotyl and leaf explants, the petioles required lower concentrations of the two growth regulators (0.44 M BA and 0.49 M IBA). Regenerated shoots could be rooted on growth regulator-free gelled full-strength MS medium. Following simple hardening procedures, the in vitro-raised plants could be transferred to soil and grown to maturity in the field.Abbreviations BA N⁶ benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige & Skoog's (1962) medium - NAA -naphthaleneacetic acid     

De novo shoot organogenesis of Pinus eldarica Medw. in vitro

Seedling-derived explants of the Afghan pine, Pinus eldarica, were cultured in a triplicate experiment to produce callus that was serially subcultured for up to three years. Callus was removed at various times and induced to regenerate shoots by de novo organogenesis. The shoot regeneration process involved the identification of four discrete developmental steps, each requiring a separate cultural manipulation. In one case a regenerated shoot was induced to root following an auxin pulse treatment. Induction and limited development of buds in callus derived from mature-tree explants was also achieved. This is the first reproducible system for shoot regeneration from long-term callus cultures of a conifer.Abbreviations MMS modified Murashige and Skoog (1962) medium - BA 6-benzylaminopurine - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA 1-naphthaleneacetic acid     

Morphoregulatory effect of plant growth regulators on Hypericum perforatum L. Seedlings

The morphogenetic response of Hypericum perforatum seedlings to different auxin and cytokinin concentrations was studied. A stimulation of the concentration-dependent rooting ability was observed under the influence of indole-3-acetic acid and indole-3-butyric acid. Rooting was not enhanced by the effects of 2,4-dichlorophenoxyacetic acid and 1-naphtaleneacetic acid. Differentiated roots were isolated and cultured in liquid media with the same combination of growth-promoting auxins. Chromosome counts in root tip cells after long-term cultivation indicated a high degree of chromosomal instability. Multiple shoot formation occurred under the influence of 6-benzylaminopurine and kinetin. Adenine and 6-(γ,γ-dimethylallylamino)-purine did not stimulate shoot differentiation. No differences in the morphogenetic response to auxins and cytokinis were detected between diploid and tetrapoloid plants.     

Plants regenerated from auxin-auxotrophic variants are inviable

Summary Shoots regenerated from auxin-auxotrophic variants of Nicotiana plumbaginifolia were inviable when cultured in vitro in the absence of auxin. Variant shoots survived longer when grafted to wild-type stocks but eventually died after a characteristic pattern of degeneration. The auxin auxotrophs were isolated after mutagen treatment by a total isolation method as infrequent variants amongst haploid protoplast-derived cell colonies. The variants responded to several active auxins but, unlike the wild type, not to cytokinin. Plant regeneration from variant cultures ceased at early stages of shoot formation after complete withdrawal of auxin from the medium.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromo-2-deoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethyl sulfoxide - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MNNG N-methyl-N-nitro-N-nitrosoguanidine - NAA 1-naphthaleneacetic acid     

Effect of plant growth regulators on somatic embryogenesis in leaf cultures of Coffea canephora

The effects of plant growth regulators on somatic embryogenesis were studied in leaf cultures of Coffea canephora. The maximum number of somatic embryos were obtained on media that contained only cytokinin as a plant growth regulator. All of the auxins tested (NAA, IBA, IAA and 2, 4-D) inhibited the formation of embryos. The optimal concentration of each cytokinin (2-iP, BA and kinetin) for somatic embryogenesis was 5 M. Under optimal conditions, each explant formed more than 100 embryoids with little callus and few adventitious roots. Embryoids were formed only at the cut edges of the leaf discs. Cytokinins were absorbed only at the cut edges of leaf discs that were in contact with the medium, and were not transported to other parts of the explant.Abbreviations 2-iP iso-pentenyladenine - BA benzyladenine - NAA 1-naphthaleneacetic acid - IBA indole-3-butyric acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Plant genotype and growth regulators interaction affecting in vitro morphogenesis of blackberry and raspberry

The morphogenic response of somatic (leaf and petiole) and de-differentiated tissue (callus) of two blackberry (Rubus fruticosus) and one raspberry (Rubus idaeus) cultivars have been studied in vitro. With the aim to induce regeneration the effect of two sets of plant growth regulator (PGR) combinations (high cytokinin/auxin ratios and high auxin/cytokinin ratios) in Murashige and Skoog basal medium, were analysed. The three cultivars were characterised by a qualitatively different morphogenic response to the PGR combinations. Raspberry adventitious shoot regeneration from somatic tissue was improved by the 6-benzylaminopurine (BAP)/indol-3-butyric acid (IBA) combinations. On the contrary, shoot regeneration of both blackberry cultivars was reduced by high concentrations of BAP and completely inhibited by BAP/IBA combination. Media supplemented with high auxin/cytokinin ratios promoted callus production and root differentiation according to genotype and type of auxin. All the genotypes responded to media supplemented with IBA. 2,4-dichlorophenoxyacetic acid induced good callus formation in blackberry, but was toxic to raspberry. Indirect shoot formation was observed only in callus of blackberry cultivar Hull Thornless cultivated on medium with 10 μM BAP, the same concentration able to trigger efficient direct shoot regeneration from leaf explants of the same cultivar. This revised version was published online in July 2006 with corrections to the Cover Date.     

Tissue,cell culture and micropropagation of Mandevilla velutina,a natural source of a bradykinin antagonist

Leaf, stem and root explants of Mandevilla velutina were cultured in vitro and produced vigorous callus in LS basal medium containing one auxin (2,4-D or NAA) plus BAP. Calli can be subcultured indefinitely with vigorous growth. Subculture of calli to NAA (1.0 mg/l) plus BAP (5.0 mg/l) caused profuse regeneration of shoots. Isolated shoots were rooted in basal medium plus NAA (5.0 mg/l) or IBA (8.0 mg/l). Rapidly growing cell suspensions can be easily obtained from friable callus cultured in liquid medium.Abbreviations LS Linsmaier & Skoog - 2,4-D 2,4 dichlorophenoxi-acetic acid - NAA -naphthalene-acetic acid - GA₃ gibberellic acid - IAA indole-3-acetic acid - BAP 6-benzylaminopurine - IBA indole-3-butyric acid     

Rapid micropropagation for Mucuna pruriens f. pruriens L.

A rapid micropropagation system was developed for Mucuna pruriens f. pruriens using explants from 1-week-old aseptically grown seedlings. Multiple shoot regeneration occurred following an initial callus growth on Revised Tobacco (RT) medium supplemented with 2.7 M NAA and 9.8 M 2iP. Maximum number of shoot regeneration was achieved only from seedling explant 6 to 7 days old. More than 90% of the regenerated shoots could be rooted on half-strength liquid RT medium supplemented with 2.7 M NAA. Plantlets readily adopted to greenhouse conditions. This system provides a new tool for micropropagation of Mucuna pruriens f. pruriens, an important medicinal plant.Abbreviations BAP 6-benzylaminopurine - Kn kinetin - 2iP 2-isopentenyladenine - AdS adenine sulphate - NAA alpha-naphthalene acetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - RT Revised Tobacco medium (Kaul and Staba, 1968)     

Plantlet regeneration from petiole explants of the african horned cucumber,Cucumis metuliferus

Plantlet regeneration in Cucumis metuliferus from several explant sources, including cotyledons, leaves, hypocotyls and petioles, was evaluated on Murashige and Skoog's medium containing various combinations of auxin (IAA, NAA, 2,4-d) and cytokinin (BA, kinetin, zeatin), Callus development was obtained within 4 to 5 weeks on all growth regulator combinations which were tested at concentrations ranging from 1.0 M to 4.0 M of each. The response was similar when the tissues were incubated under light or in continuous darkness. Differentiation of callus to form adventitious buds or shoot primordia occurred only with petiole explants on medium containing NAA/BA or 2,4-d/BA at 2.0/1.0 M; none of these calluses, however, differentiated further to form shoots. When the differentiated calluses derived from petiole explants which had been initiated on 2,4-d/BA at 2.0/1.0 M were transferred onto medium with 2.0 M zeatin, formation of shoots occurred within 2 to 3 weeks. The frequency of shoot formation was 14.6%. Subculture of these shoots onto MS medium without growth regulators gave rise to plantlets of normal appearance. Regeneration in C. metuliferus requires callus initiation on an appropriate growth regulator regime followed by transfer to a medium containing the cytokinin, zeatin, and can be achieved within 10–12 weeks.Abbreviations BA 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA napthaleneacetic acid     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of <Emphasis Type="Italic">Rhododendron wattii</Emphasis> Cowan—a critically endangered and endemic plant from India

Rhododendron wattii Cowan is a rare and endangered plant found in northeast India. In an effort to boost specimen numbers, experiments of in vitro seed germination, shoot induction on different media supplemented with the cytokinin isopentenyladenine (2iP), and root induction with auxins α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) in woody plant medium (WPM) were carried out. A maximum mean shoot number of 7.72 per explant were obtained from nodal explants cultured on WPM and 39.36 μM 2iP with a maximum mean shoot length of 2.30 cm per explant. Among the auxins investigated for root induction, IBA at 2.45 μM was found to produce the most and the longest roots, when compared to other treatments. However, WPM supplemented with 0.2% (w/v) activated charcoal also showed 100% root formation with shoots having broader leaves compared to auxin treatments. About 60% of in vitro rooted plantlets transferred from lab to greenhouse conditions survived. Sixty acclimatized plants were reintroduced in the vicinity of their natural habitat at Naga Heritage Village, Kisama, Nagaland, in May 2016 for ex situ conservation. Survival of the reintroduced plants was confirmed during the field visit conducted in November 2016.     

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing -naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.Abbreviations BAP 6-benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kn Kinetin - MS Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid     

Auxin structure and activity on tomato morphogenesis in vitro and pea stem elongation

In order to understand better the relationship between auxin structure and activity on morphogenesis and cell elongation, six different auxins were tested on the regeneration of tomato (Lycopersicon esculentum Miller var. Alice) from cotyledons and on pea (Pisum sativum L. var. Alaska) stem elongation. The auxins were: indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), 1, 2-benzisoxazole-3-acetic acid (BOA), 1,2-benzisothiazole-3-acetic acid (BIA), 1-naphthalenacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D). All these compounds obey the minimum requirement rules for auxin activity and all were effective on cell elongation. At the dose of 10 M and in the absence of cytokinin, they all, except 2,4-D, induced roots, while in the presence of cytokinin they induced shoots, roots, hairy root-like filaments (HRLF) or callus depending on their concentration. The morphogenetic pattern did not change by varying cytokinin concentration. We conclude that auxin structure plays a minor role in morphogenesis or cell elongation, because it is only responsible for variations in the level of auxin activity.     

Formation in vitro of ripe tomato fruits from thin layer explants of flower pedicels

Thin longitudinal sections cut from pedicels of fifteen cultivars of tomato (Lycopersicon esculentum) were grown in vitro on Murashige-Skoog medium supplemented with various concentrations of different auxins and cytokinins. Isatin (an auxin precursor slowly converted to an active auxin) was the most effective source of auxin for the formation of buds without prior root formation, while zeatin was the most effective cytokinin for growth and development of the buds. Flower buds and ripe fruits developed consistently from explants of the cultivar Pixie Hybrid II treated with 10 M isatin plus 3 M zeatin as the cytokinin. Fruits developed parthenocarpically, grew to a diameter of about 15 mm, ripened promptly, and possessed normal color and flavor.Abbreviations BAP benzylaminopurine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA isopentyladenosine - NAA -napthaleneacetic acid     

Plant regeneration through the direct induction of shoot buds from petiole explants of Jatropha curcas: a biofuel plant

An efficient and reproducible method for the regeneration of Jatropha curcas plants has been developed. The method employed direct induction of shoot buds from petiole explants, without the formation of an intervening callus using a Murashige and Skoog (MS) medium supplemented with different concentrations of thidiazuron (TDZ). The best induction of shoot buds (58.35%) and the number of shoot buds per explant (10.10) were observed when in vitro petiole explants were placed horizontally on MS medium supplemented with 2.27 µM TDZ after 6 weeks. The induced shoot buds were transferred to MS medium containing 10 µM kinetin (Kn), 4.5 µM 6-benzyl aminopurine (BAP) and 5.5 µM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with different concentrations and combinations of BAP, indole-3-acetic acid (IAA), NAA and indole-3-butyric acid (IBA). MS medium supplemented with 2.25 µM BAP and 8.5 µM IAA was found to be the best combination for shoot elongation and 3.01–3.91 cm elongation was achieved after 6 weeks. However, significant differences in plant regeneration and shoot elongation were observed among the genotypes studied. The orientation (horizontal or vertical) and source (in vitro or in vivo) of explants also significantly influenced plant regeneration. The elongated shoots could be rooted on half-strength MS medium supplemented with 2% sucrose, different concentrations and combinations of IBA, IAA and NAA, and 0.25 mg L⁻¹ activated charcoal. Half-strength MS medium supplemented with 2% sucrose, 15 µM IBA, 5.7 µM IAA, 5.5 µM NAA and 0.25 mg L⁻¹ activated charcoal was found to be the best for promoting rooting. The rooted plants could be established in soil with more than 90% survival.