Somatic embryogenesis and direct as well as indirect organogenesis in Lilium pumilum DC. Fisch., an endangered ornamental and medicinal plant

Somatic embryogenesis and organogenesis in Lilium pumilum were successfully regulated by picloram, α-naphthaleneacetic acid (NAA), and 6-benzyladenine (BA). In organogenesis, the highest shoot regeneration frequency (92.5%) was obtained directly from bulb scales on Murashige and Skoog (MS) medium containing 2.0 mg L^?1 BA and 0.2 mg L^?1 NAA, while organogenic callus (OC) formed from leaves on MS medium supplemented with 1.0 mg L^?1 BA and 0.5 mg L^?1 NAA. Following subculture, 76.7% of OC regenerated shoots. In somatic embryogenesis, the combination of picloram and NAA increased the amount of embryogenic callus (EC) that formed with a maximum on 90.7% of all explants which formed 11 somatic embryos (SEs) per explant. Differences between EC and OC in cellular morphology and cell differentiation fate were easily observed. SEs initially formed via an exogenous or an endogenous origin. The appearance of a protoderm in heart-shaped SE and the bipolar shoot–root development in oval-shaped SE indicated true somatic embryogenesis. This protocol provides a new and detailed regulation and histological examination of regeneration pattern in L. pumilum.     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

In vitro propagation of <Emphasis Type="Italic">Psoralea corylifolia</Emphasis> L. by somatic embryogenesis in cell suspension culture

An effective protocol was developed for in vitro propagation of Psoralea corylifolia via somatic embryogenesis in cell suspension culture. Embryogenic callus was obtained on Murashige and Skoog (MS) medium supplemented with 6 μM naphthaleneacetic acid (NAA) and 30 μM glutamine from transverse TCLs from 10-day-old hypocotyl explants with a 96.4% frequency. Embryogenic callus produced a higher number of somatic embryos (123.7 ± 1.24 per gram fresh weight callus) on MS medium containing 30 g l^?1 sucrose, 1 μM NAA, 4 μM benzyladenine (BA), 15 μM glutamine and 2 μM abscisic acid (ABA) after 4 weeks of culture. Somatic embryos successfully germinated (97.6%) on ½ MS medium containing 20 g l^?1 sucrose, 8 g l^?1 agar and supplemented with 2 μM BA, 1 μM ABA and 2 μM gibberellic acid (GA₃) within 2 weeks of culture. Somatic embryos developed into normal plants, which hardened with 100% efficiency in soil in a growth chamber. Plants were successfully transferred to greenhouse and subsequently established in the field. Plant survival percentage in the field differed with seasonal variations. Average psoralen content of 12.9 μg g^?1 DW was measured in different stages of somatic embryo development by high-performance liquid chromatography (HPLC). This protocol will be helpful for efficient propagation of elite clones on a mass scale, conservation efforts of this species and for secondary metabolites production studies.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of <Emphasis Type="Italic">Melothria maderaspatana</Emphasis> via indirect organogenesis

An effective protocol was developed for in vitro regeneration of the Melothria maderaspatana via indirect organogenesis in liquid and solid culture systems. Organogenesis was achieved from liquid culture calluses derived from leaf and petiole explants of mature plants. Organogenic calluses (98.2?±?0.36 and 94.8?±?0.71%) were induced from both leaf and petiole explants on Murashige and Skoog (MS) liquid medium containing 6.0 µM 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 µM thidiazuron (TDZ); and 6.0 µM 2,4-D and 1.0 µM benzyladenine (BA) combinations, respectively. Adventitious shoot regeneration (68.2?±?0.06 shoots per explant) was achieved on MS medium supplemented with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water and 0.06 mM glutamine from leaf-derived calluses. Petiole-derived calluses produced adventitious shoots (45.4?±?0.09 shoots per explant) on MS medium fortified with 2.0 µM BA, 4.0 µM TDZ, 10% v/v coconut water, and 0.08 mM glutamine. Elongation of shoots occurred in MS medium with 2.0 µM gibberellic acid (GA₃). Regenerated shoots (2–3 cm in length) rooted (74.2?±?0.38%) and hardened (85?±?1.24%) when they were transferred to 1/2-MS medium supplemented with 3.0 µM indole-3-butyric acid (IBA) followed by garden soil, vermiculate, and sand (2:1:1 ratio) mixture. The elongated shoots (4–5 cm in length) were exposed simultaneously for rooting as well as hardening (100%) in moistened [(1/8-MS basal salt solution with 5 µM IBA and 100 mg l^?1 Bavistin® (BVN)] garden soil, vermiculate, and sand (2:1:1 ratio) mixture. Subsequently, the plants were successfully established in the field. The survival percentage differed with seasonal variations.     

Recurrent somatic embryogenesis and plant regeneration from seedlings of <Emphasis Type="Italic">Hepatica nobilis</Emphasis> Schreb.

A method for secondary somatic embryogenesis was developed on embryos derived from embryogenic callus formed on Hepatica nobilis seedlings. Somatic embryogenesis (SE) was induced on seedlings (on the hypocotyl and epicotyl parts) grown on the Murashige and Skoog (1962) medium (MS) supplemented with 1 µM naphthaleneacetic acid (NAA), and/or 0.1 µM 6-benzyladenine (BA) and on medium without plant growth regulators (PGR). The best response of embryogenic callus formation was observed on the medium containing 1 µM NAA alone or with 0.1 µM BA. Individual somatic embryos, formed on embryogenic callus on the medium without PGR (MS0), at heart, torpedo and cotyledonary stage, were transferred to the media where secondary somatic embryo formation and development into plantlets occurred. Although the most efficient repetitive cycles of secondary SE were recorded for all stages of somatic embryos (heart, torpedo, cotyledonary) on the MS0 medium (77.8–87.4 %), secondary somatic embryos were also obtained on all media supplemented with cytokinins. The best rate of somatic embryos germination was achieved on MS media with 0.2 µM NAA and 2 µM BA, and 0.1 µM NAA and 1 µM BA (48.8–52.0 %) when more mature embryos (cotyledonary stage) were used. Plantlets grown from somatic embryos were successfully acclimatized to greenhouse conditions.     

Differential responses to somatic embryogenesis of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.)

To assess the potential of different genotypes of Brazilian oil palm (Elaeis guineensis Jacq.) to somatic embryogenesis and somatic embryo proliferation, mature zygotic embryos of nine commercial genotypes of E. guineensis (BRSC2001, BRSC2328, BRSC2301, BRSC3701, BRSCM1115, BRSC7201, BRSC2528, BRSC2501, and BRSCN1637) were used. Explants were incubated on Murashige and Skoog (MS) supplemented with 450 μM picloram, 3.0 % sucrose, 500 mg l^?1 glutamine, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. After induction, for differentiation and maturation, the embryogenic calli (ECs) were transferred into fresh medium supplemented with 0.6 μM naphthaleneacetic acid (NAA) and 12.30 μM 2-isopentenyladenine (2iP) or 40 μM picloram in combination with 0.3 g l^?1 activated charcoal, and 500 mg l^?1 glutamine. Somatic embryos were converted into plants on MS medium with macro- and micro-nutrients at half strength, 2 % sucrose, and 2.5 g l^?1 activated charcoal, and gelled with 2.5 g l^?1 Phytagel. In general, zygotic embryos swelled after 14 days. Primary calli, which were observed in all the genotypes after 45–60 days of culture, eventually progressed to ECs at 90 days. At this time, scanning electron microscopy (SEM) analysis showed cellular differences between compact and friable calli. After 150 days in the induction phase, the ECs with proembryos that were transferred to the medium for differentiation and maturation, differentiated asynchronically into somatic embryos at globular and torpedo stages. The results showed that BRSC2328 and BRSCM1115 had the highest potential for EC formation (90–100 %) and somatic embryo differentiation (40.7 and 52.5 somatic embryos per callus, respectively) when compared to other genotypes. After approximately 90 days of culture on MS basal medium without growth regulators, protrusion of the leaf primordia was observed, characterizing the onset of germination of the somatic embryos into plants.     

Direct somatic embryogenesis of potato [<Emphasis Type="Italic">Solanum tuberosum</Emphasis> (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO₃, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO₃ for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (>?174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.     

Recovery of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) plants transformed with the antifungal plant defensin gene PDF1.2

Embryogenic avocado cultures derived from ‘Hass’ protoplasts were genetically transformed with the plant defensin gene (pdf1.2) driven by the CaMV 35S promoter in pGPTV with uidA as a reporter gene and bar, the gene for resistance to phosphinothricin, the active ingredient of the herbicide Finale® (Basta) (Bayer Environmental Science, Research Triangle Park, Durham, NC ). Transformation was mediated by Agrobacterium tumefaciens strain EHA105. Transformed cultures were selected in the presence of 3.0 mg l^?1 phosphinothricin in liquid maintenance medium for 3–4 mo. Liquid maintenance medium consisted of modified MS medium containing (per liter) 12 mg NH₄NO₃ and 30.3 mg KNO₃ and supplemented with 0.1 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 30 g l^?1 sucrose, 3.0 mg l^?1 phosphinothricin, and 0.41 μM picloram. Somatic embryo development from transformed cultures was initiated on MS medium supplemented with 45 g l^?1 sucrose, 4 mg l^?1 thiamine HCl, 100 mg l^?1 myo-inositol, 10% (v/v) filter-sterilized coconut water, 3.0 mg l^?1 phosphinothricin, and 6.0 g l^?1 gellan gum. Limited plant recovery occurred from somatic embryos on semi-solid MS medium supplemented with 3.0 mg l^?1 phosphinothricin, 4.44 μM 6-benzylaminopurine (BA), and 2.89 μM GA₃; transformed shoots were micrografted on in vitro-grown seedling rootstocks. Approximately 1 yr after acclimatization in the greenhouse, transformed shoots were air-layered to recover transformed roots. Genetic transformation of embryogenic cultures, somatic embryos, and regenerated plants was confirmed by polymerase chain reaction (PCR), Southern blot hybridization, the XGLUC reaction for uidA, and application of the herbicide Finale® to regenerated plants.     

Elicitor mediated enhancement of wedelolactone in cell suspension culture of <Emphasis Type="Italic">Eclipta alba</Emphasis> (L.) Hassk

The present research focused on enhancing the production of wedelolactone through cell suspension culture (CSC) in Eclipta alba (L.) Hassk. With an aim of attaining a sustainable CSC, various plant growth regulators, elicitors and agitation speed were examined. Nodal segments of in vitro propagated plantlets induced the maximum percentage (93.47?±?0.61%) of callus inoculated on Murashige and Skoog (MS) medium fortified with picloram (2 mg L^?1). The growth kinetics of CSC exhibited a sigmoid pattern with a lag phase (0–6 days), a log phase (6–18 days), a stationary phase (18–24 days) and then death phase thereafter. The highest biomass accumulation in CSC with 7.09?±?0.06 g 50 mL^?1 fresh weight, 1.52?±?0.02 g 50 mL^?1 dry cell weight, 1.34?±?0.01?×?10⁶ cell mL^?1 total cell count and 57.00?±?0.58% packed cell volume was obtained in the liquid MS medium supplemented with 1.5 mg L^?1 picloram plus 0.5 mg L^?1 kinetin at 120 rpm. High performance thin layer chromatography confirmed that yeast extract (biotic elicitor) at 150 mg L^?1 accumulated more CSC biomass with 1.22-fold increase in wedelolactone (288.97?±?1.94 µg g^?1 dry weight) content in comparison to the non-elicited CSC (237.78?±?0.04 µg g^?1 dry weight) after 120 h of incubation. Contrastingly, methyl jasmonate (abiotic elicitor) did not alter the biomass but increased the wedelolactone content (259.32?±?1.06 µg g^?1 dry weight) to an extent of 1.09-fold at 100 µM. Complete plantlet regeneration from CSC was possible on MS medium containing N⁶-benzyladenine (0.75 mg L^?1) and abscisic acid (0.5 mg L^?1). Thus, the establishment of protocol for CSC constitutes the bases for future biotechnological improvement studies in this crop.     

Optimization of culture conditions for the production of polysaccharides and kinsenoside from the rhizome cultures of <Emphasis Type="Italic">Anoectochilus roxburghii</Emphasis> (Wall.) Lindl.

The present study designed two sets of experiments by using the uniform design method and investigated the effects of medium components on the accumulation of bioactive compounds (polysaccharide and kinsenoside) in rhizomes, in order to select a suitable culture medium for the rhizome suspension culture of Anoectochilus roxburghii (Wall.) Lindl. Among the combinations of Murashige and Skoog (MS) medium strengths and plant growth regulator (benzylaminopurine, BA; kinetin, KT; and α-naphthaleneacetic acid, NAA) concentrations, and the combinations of nitrogen, phosphorus, and sucrose concentrations, the maximum yield of polysaccharides and kinsenoside was achieved with 0.75 × MS?+?2.0 mg L^?1 BA?+?0.2 mg L^?1 KT?+?0.5 mg L^?1 NAA and 45 mM nitrogen?+?0.93 mM phosphorus?+?35 g L^?1 sucrose, respectively. Therefore, the optimal rhizome suspension culture medium was 0.75 × MS medium supplemented with 2.0 mg L^?1 BA, 0.2 mg L^?1 KT, 0.5 mg L^?1 NAA, and 35 g L^?1 sucrose. Yeast extract (YE) enhanced bioactive compound accumulation in rhizomes. The polysaccharide and kinsenoside production was significantly improved when 75 mg L^?1 YE was added to the culture medium after 30 d of rhizome suspension culture; 8.3 g L^?1 of polysaccharide and 6.1 g L^?1 of kinsenoside were obtained after 4 d of YE treatment. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity of YE-treated rhizomes was higher than that of YE-untreated rhizomes, demonstrating enhanced antioxidant activity of the treated bioreactor-cultured rhizomes.     

Micropropagation of mature <Emphasis Type="Italic">Quercus ilex</Emphasis> L. trees by axillary budding

This paper reports the successful micropropagation of mature Quercus ilex trees known as reluctant to in vitro propagation. Crown branch segments collected from 30 to 100 year-old trees were forced in order to promote the production of sprouting shoots that were used as a source of explants for initiating the cultures. Sterilization was critical and required low-level disinfestation protocols. Six out of the eight mature genotypes attempted were successfully inoculated and then maintained in culture with varying responses. Shoot proliferation of holm oak was influenced by BA concentration, with improved multiplication and shoot appearance when the BA concentration was sequentially reduced over the culture period. Micropropagation by axillary budding was achieved by culturing shoots on a sequence of cytokinin-enriched Lloyd and McCown (WPM) media alternating 2 week-long subcultures on 0.44 µM benzyadenine (BA) first, followed by 0.22 µM BA, then 0.044 µM BA plus 0.46 µM zeatin. Sucrose concentration and agar brand affected shoot proliferation, and the best results were obtained on WPM medium supplemented with 8 g L^?1 Sigma agar (A-1296; Sigma-Aldrich) and 30 g L^?1 sucrose. Addition of 20 µM silver thiosulphate had a significant positive effect on the appearance and development of shoots with a higher number of shoots being healthy and showing reduced shoot tip necrosis and early senescence of leaves. The 18.8% of the microshoots obtained for one clone could be rooted within 15 days on a half-strength Murashige and Skoog medium containing 14.8 µM or 24.6 µM indole-3-butyric acid and 0.54 µM α-naphthalene acetic acid.     

New approaches to improve the efficiency of somatic embryogenesis in oil palm (Elaeis guineensis Jacq.) from mature zygotic embryos

We developed an efficient and simple system for inducing somatic embryogenesis and regenerating plantlets from mature zygotic embryos of oil palm. Embryogenic calli were induced from mature zygotic embryos of oil palm on modified Murashige and Skoog medium with 2,4-dichlorophenoxyacetic acid or picloram, alone or in combination with activated charcoal. The greatest frequency of embryogenic callus induction (97.5%) was obtained by culturing mature zygotic embryos on callus induction medium with 450 μM picloram and 2.5 g?L^?1 activated charcoal. Embryogenic calli proliferated on a medium with a reduced concentration of picloram. Embryogenic calli were then subcultured on a medium supplemented with 12.3 μM 2-isopentenyladenine and 0.54 μM naphthaleneacetic acid, with subcultures at 4-wk intervals. Somatic embryos were regenerated on a medium with Murashige and Skoog macro- and micronutrients at half-strength concentrations supplemented with 20 g?L^?1 sucrose, 2.5 g?L^?1 activated charcoal, and 2.5 g?L^?1 Phytagel. Detailed histological analysis revealed that somatic embryogenesis followed an indirect pathway. Primary calli were observed after 4–6 wk of culture and progressed to embryogenic calli at 12 wk. Embryogenic cells exhibited dense protoplasm, a high nucleoplasmic ratio, and small starch grains. Proembryos, which seemed to have a multicellular origin, formed after 16–20 wk of culture and successive cell divisions. Differentiated somatic embryos had a haustorium, a plumule, and the first and second foliar sheaths. In differentiated embryos, the radicular protrusion was not apparent because it generally does not appear until after the first true leaves emerge.     

Optimized culture medium for the production of flavonoids from <Emphasis Type="Italic">Orostachys cartilaginea</Emphasis> V.N. Boriss. callus cultures

The optimal culture medium for the production of flavonoid compounds from Orostachys cartilaginea V. N. Boriss. calluses was studied. In callus cultures of O. cartilaginea, the flavonoid monomer content, in decreasing order was kaempferol-3-O-rutinoside (Kp-3-rut), quercetin 3-O-glucoside (Qc-3-glc), epicatechin gallate (Ecg), kaempferide (Ke), and quercetin (Qc). The results of the uniform design experiment indicated that the production of Qc, Ke, Qc-3-glc, Kp-3-rut, and total flavonoids were satisfactory in callus grown on full salt strength (1×) of Murashige and Skoog (MS) medium supplemented with 3.5 mg L^?1 6-benzylaminopurine (BA) and 0.1 mg L^?1 1-naphthalene acetic acid (NAA). By contrast, only Ecg was found in callus grown on 0.75× MS medium supplemented with 1.5 mg L^?1 BA and 0.3 mg L^?1 NAA. A phosphate concentration of 1.25 mM in the MS medium favored the production of Qc and Ke, whereas 0.75 mM phosphate was optimal for the production of Ecg, Qc-3-glc, Kp-3-rup, and total flavonoids. The NH₄ ⁺/NO₃ ^? ratios of 30/30 mM in the MS medium promoted Ke, Ecg, Qc-3-glc, Kp-3-rup, and total flavonoid production. However, a NH₄ ⁺/NO₃ ^? ratio of 20/40 mM enhanced Qc production. The effect of sucrose concentrations on the accumulation of different flavonoid monomers was comparatively more regular. The flavonoid content increased as the sucrose concentration increased from 20 to 40 g L^?1, peaked at 40 g L^?1, and decreased at concentrations greater than 40 g L^?1. Therefore, 40 g L^?1 sucrose was optimal for the production of the five flavonoid monomers and total flavonoids. The present findings demonstrate the possibility of producing flavonoid compounds from O. cartilaginea callus.     

Somatic embryogenesis and plantlet regeneration from mature zygotic embryos of Manchurian ash (Fraxinus mandshurica Rupr.)

Using mature cotyledonary explants of Fraxinus mandshurica, an efficient plant regeneration system was developed via somatic embryogenesis. More than 67 % of mature cotyledons of zygotic embryos yielded 23–159 somatic embryos (SEs) per explant when incubated on medium consisting of half-strength Murashige and Skoog (MS) salts and vitamins (MS1/2) supplemented with 8.88 μM 6-benzyladenine (BA), 26.84 μM naphthaleneacetic acid (NAA), 75 g L^?1 sucrose, and 400 mg L^?1 casein hydrolysate (CH). Approximately, 82 % of induced SEs were observed on browning cotyledonary explants. Histological studies of cotyledon explants at various stages of somatic embryogenesis revealed that the SEs originated from single epidermal cells and developed to the globular, heart, torpedo, and cotyledonary stage embryos. Secondary somatic embryos (SSEs) formed on the surface of radicle tips of the SEs. Addition of low concentrations of NAA and 200–400 mg L^?1 CH to MS1/2 medium increased SSE induction. Cotyledonary SSEs were cultured on MS1/2 medium with 10 mM abscisic acid in the presence of light to promote maturation, and >92 % of mature SSEs were able to germinate with normal shoots. After 8 weeks in culture in the presence of light on medium with one-third of the MS macroelements as well as 0.06 μM NAA, >94 % of the germinated SSEs converted into plantlets. Plantlets acclimatized successfully to ex vitro conditions and developed normal phenotypes under field conditions.     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

<Emphasis Type="Italic">In vitro</Emphasis> plant regeneration and cryopreservation of <Emphasis Type="Italic">Arachis glabrata</Emphasis> (Fabaceae) using leaflet explants

Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L^?1 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L^?1 TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.     

An improved protocol for somatic embryogenesis and plant regeneration in macaw palm (Acrocomia aculeata) from mature zygotic embryos

An improved protocol for plant regeneration via somatic embryogenesis was developed using mature macaw palm (Acrocomia aculeata) zygotic embryos as initial explant. For induction of the embryogenic callus (EC), two basic media (BM) were tested consisting of Murashige and Skoog and Eeuwens (Y3) salts with 30 g L^?1 sucrose, 0.5 g L^?1 glutamine and 2.5 g L^?1 Phytagel. The 3,6-dichloro-2-methoxybenzoic acid (dicamba), 4-amino-3,5,6-trichloro-picolinic acid (picloram) and 2,4-dichlorophenoxyacetic acid (2,4-D) auxins were added to the culture media at concentrations of 0, 1.5 or 3.0 mg L^?1. After 240 days, the embryogenic calli were transferred to the respective BM media with auxin concentrations reduced to 0.5 or 1.0 mg L^?1 in order to differentiate the somatic embryos (SEs). Plant regeneration was performed on the BM media without growth regulators. Embryogenic calli were observed after 180 days of culture and in all treatments with auxin. The Y3 medium showed the best EC formation results (60.8 %). These calli showed yellowish coloration, compact consistency and nodular aspect. After 60 days in differentiation medium, SEs were verified in different stages of development. Histological analysis showed that the SEs were formed from a nodular EC. The SEs generally presented unicellular origin with suspensor formation, and at the end of development, bipolar embryos were observed. The plant regeneration frequency reached levels up to 31.9 % when using induction medium consisting of Y3 associated to 1.5 mg L^?1 of 2,4-D and the subsequent auxin reduction to 0.5 mg L^?1 in the differentiation stage. Regenerated plants showed normal development, with root and aerial part growth.     

Somatic embryogenesis in banana (Musa acuminata AAA cv. Grand Naine): effect of explant and culture conditions

An improved, rapid, reproducible, and simple protocol has been developed for somatic embryogenesis in banana cv. ‘Grand Naine’ using explants derived from actively growing multiple shoot cultures. Many restrictive factors remain in banana embryogenesis such as long duration, unpredictability, and a high degree of genotype dependence. In the present study, we used split shoot tips from 4-wk-old cultures as explants. Somatic embryos were induced in 15 d directly in Murashige and Skoog (MS) medium supplemented with different combinations of 0–8.28 μM picloram and 0.22–4.44 μM 6-benzylaminopurine (BA) without callus formation. Maximum embryo induction (100%) occurred when 4.14 μM picloram and 0.22 μM BA were used. Conversion of somatic embryos into plantlets occurred sporadically (2–3%) in MS medium containing α-naphthalene acetic acid (NAA; 0.53–2.68 μM) together with BA (2.22–44.39 μM), or thidiazuron (4.54 μM) plus glutamine (200 mg/L). This protocol is far superior to those already reported for fast and high frequency induction of somatic embryo. In liquid agitated culture, individual embryos separated easily and produced a large number of secondary embryos within 10 d which, upon transfer to filter paper overlaid on MS liquid medium supplemented with 4.44 μM BA, resulted in conversion (3%) into plantlets.     

Somatic embryogenesis and plant regeneration of blackberry using the thin cell layer technique

Limonium ‘Misty Blue’ is an interspecific hybrid of Limonium latifolium and L. bellidifolium and has a huge demand in floriculture business as both fresh and dry flowers with stunning purple-blue blooms. The propagation only through vegetative means restrict the popularization of this plant to the flower growers. We therefore optimized an efficient micropropagation protocol for direct organogenesis from root explants, as leaf is not conducible to respond in culture. 61.43% of root explants directly formed shoot buds on their surface after 4-weeks of culture in media containing ½ MS, 43.82 mM sucrose 2.22 µM BA and 1.07 µM NAA. The shoot buds failed to differentiate into healthy shoots unless the previous medium was replaced by full strength MS, and 87.64 mM sucrose along with 0.44 µM BA and 1.07 µM NAA. Encapsulations of juvenile shoots were carried out by 3% sodium alginate and 100 mM CaCl₂ which were again successfully stored at 4?°C for 30 days along with 56.79% of plant recovery in MS?+?0.44 µM BA?+?4.5 µM IBA?+?87.64 mM sucrose containing medium. 150 synthetic seed derived full grown plants were successfully acclimatized in green house, where a total of 101 plants survived after secondary hardening. The ISSR analysis revealed genetic homogeneity of synthetic seed derived hardened plants.