Promotion of shoot regeneration of <Emphasis Type="Italic">Swertia chirata</Emphasis> by biosynthesized silver nanoparticles and their involvement in ethylene interceptions and activation of antioxidant activity

A complete protocol for the in vitro induction of Eclipta alba tetraploids has been optimized to enhance the wedelolactone content, an anti-cancerous compound. The effects of different concentrations of colchicine (0, 0.01, 0.05, 0.1, 0.2 and 0.3%; w/v) along with treatment durations (12, 24, 36 and 48 h) were investigated on shoot tip (ST) and nodal segment (NS). The treated explants were then incubated on Murashige and Skoog (MS) medium having 1.5 mg L^?1 N⁶-benzylaminopurine and 0.5 mg L^?1 α-napthalene acetic acid for shoot regeneration and afterward root was induced on 1.0 mg L^?1 indole-3-acetic acid enriched ½MS medium. The tetraploids of E. alba were proficiently induced by the treatment of 0.1% colchicine for 24 h. The highest tetraploid induction efficiency was obtained from ST (30.56%) in comparison to the NS (22.22%). Analysis by spectrophotometry and flow cytometry showed that colchicine treated plants contained higher quantity of DNA than diploid plants. Cytological studies demonstrated doubled the chromosome number in tetraploids (2n?=?4x?=?44) than diploids (2n?=?2x?=?22). The ploidy level enhancement lead to alteration of other traits, like increased plant height, stem diameter, leaf size, stomatal size and chlorophyll content. As determined through high performance thin-layer chromatography, the ultimate achievement of this technique is the higher accumulation of wedelolactone in tetraploid plants (300.32 µg g^?1 dry weight) in evaluation to in vitro diploid (131.31 µg g^?1 dry weight) and in vivo diploid mother plants (93.26 µg g^?1 dry weight), thus improving the pharmaceutical value of E. alba.     

Effect of over-expression of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> PINORESINOL LARICIRESINOL REDUCTASE (<Emphasis Type="Italic">LuPLR</Emphasis>) gene in transgenic <Emphasis Type="Italic">Phyllanthus amarus</Emphasis>

Shoot tip explants of Phyllanthus amarus were cocultivated with Agrobacterium tumefaciens strain LBA 4404 carrying plasmid pCAMBIA 2301 harbouring genes coding for betaglucuronidase (gus), kanamycin (kan), and neomycin phosphotransferase II (nptII) along with a gene coding for Linum usitatissimum PINORESINOL LARICIRESINOL REDUCTASE (Lu-PLR). Transformed shoot tip explants were maintained in a Murashige and Skoog (MS) medium containing TDZ 1.54 mg l^?1, kan 50 mg l^?1 and cephotaxime 62.5 mg l^?1. The optimum medium for regeneration of multiple shoots was MS supplemented with TDZ 1.54 mg l^?1, kan 50 mg l^?1. Efficient and effective rooting of plantlets was achieved by culturing the in vitro regenerated shoots on liquid ½ MS medium containing 0.7 mg l^?1 indole 3-butyric acid (IBA) and 5 mg l^?1 kan. Rooted plants were acclimatized in the mixtures of vermiculite and soil. The transformation of kan-resistant plantlets regenerated from shoot-tip explants was confirmed by GUS and polymerase chain reaction (PCR) analysis. Southern blot and reverse transcribed PCR (RT-PCR) analysis confirmed successful integration and expression of Lu-PLR gene. Quantitative analysis of phyllanthin performed on transgenic and wild plants using high-performance liquid chromatography (HPLC) revealed that transgenic lines contained higher phyllanthin content (0.3–0.81% w/w) than wild plants (0.09% w/w). The highest yield of phyllanthin was detected in transgenic lines was up to 1.16, 1.22 and 1.23 folds higher than that of wild plant. This report highlights the transgenic approach to enhance the contents of phyllanthin and hypophyllanthin.     

Efficient transformation of <Emphasis Type="Italic">Actinidia arguta</Emphasis> by reducing the strength of basal salts in the medium to alleviate callus browning

An efficient transformation system for high-throughput functional genomic studies of kiwifruit has been developed to overcome the problem of necrosis in Actinidia arguta explants. The system uses Agrobacterium tumefaciens strain EHA105 harbouring the binary vector pART27-10 to inoculate leaf strips. The vector contains neomycin phosphotransferase (nptII) and β-glucuronidase (GUS) (uidA) genes. A range of light intensities and different strengths of Murashige and Skoog (MS) basal salt media was used to overcome the problem of browning and/or necrosis of explants and calli. Callus browning was significantly reduced, resulting in regenerated adventitious shoots when the MS basal salt concentration in the culture medium was reduced to half-strength at low light intensity (3.4 μmol m^?2 s^?1) conditions. Inoculated leaf strips produced putative transformed shoots of Actinidia arguta on half-MS basal salt medium supplemented with 3.0 mg l^?1 zeatin, 0.5 mg l^?1 6-benzyladenine, 0.05 mg l^?1 naphthalene acetic acid, 150 mg l^?1 kanamycin and 300 mg l^?1 Timentin^®. All regenerated plantlets were deemed putative transgenic by histochemical GUS assay and polymerase chain-reaction analysis.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation,micromorphological studies and <Emphasis Type="Italic">ex vitro</Emphasis> rooting of cannon ball tree (<Emphasis Type="Italic">Couroupita guianensis</Emphasis> aubl.): a multipurpose threatened species

In vitro propagation methods using seeds and nodal segments of a 21-year old Couroupita guianensis - a medicinally important but threatened tree have been developed. Hundred percent of the seeds germinated on half strength Murashige and Skoog (MS) medium with 2.0 mg l^?1 indole-3 butyric acid (IBA). Nodal segments were found most suitable for the establishment of cultures. About 90 % explants responded and 4.1 ± 0.23 shoots per node were induced after five weeks of inoculation on MS medium +4.0 mg l^?1 6-benzylaminopurine (BAP). Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro produced shoots on fresh medium. Maximum number (8.2 ± 0.17) of shoots were regenerated on MS medium with 1.0 mg l^?1 each of BAP and Kinetin (Kin) + 0.5 mg l^?1 α-naphthalene acetic acid (NAA) with additives (50 mg l^?1 of ascorbic acid and 25 mg l^?1 each of adenine sulphate, L-arginine and citric acid). The multiplied shoots rooted (4.3 ± 0.26 roots/shoot) on half strength MS medium with 2.5 mg l^?1 IBA. All the shoots were rooted ex vitro when pulse treated with 400 mg l^?1 of IBA for five min with an average of 7.3 ± 0.23 roots per shoot. Nearly 86 % of these plantlets were acclimatized within 7–8 weeks and successfully transferred in the field. Biologically significant developmental changes were observed during acclimation particularly in leaf micromorphology in terms of changes in stomata, veins and vein-islets, and trichomes. This study helps in understanding the response by the plants towards outer environmental conditions during acclimatization. This is the first report on micropropagation of C. guianensis, which could be used for the large-scale multiplication, restoration and conservation of germplasm of this threatened and medicinally important tree.     

Establishment of long-term proliferating shoot cultures of elite <Emphasis Type="Italic">Jatropha curcas</Emphasis> L. by controlling endophytic bacterial contamination

Leaf explants of the second or third node were collected from field-grown elite Jatropha curcas trees and incubated in Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) medium supplemented with growth regulators. Direct shoot organogenesis was induced when explants were incubated in a medium containing 0.5 mg l^?1 benzyladenine (BA) and 0.1 mg l^?1 indolebutyric acid (IBA). A maximum of seven shoot buds differentiated within 6 weeks of culture incubation. Indirect shoot organogenesis was obtained when explants were incubated in the medium supplemented with 0.5 mg l^?1 BA along with 1.0 mg l^?1 each of 2,4-dichlorophenoxyacetic acid (2,4-D) and indoleacetic acid (IAA). A pulse treatment of 0.5 mg l^?1 thidiazurone (TDZ) and 0.1 mg l^?1 IBA for 5 days was necessary for shoot organogenesis in green compact callus before subculture into 0.5 mg l^?1 BA and 0.1 mg l^?1 IBA containing medium. Leaf explants of J. curcas, collected from the field, contained endophytic bacterial contamination, which expressed itself after 2–3 subcultures. These bacteria were cultured and identified as Enterobacter ludwigii. After staining, these were found as gram-negative bacteria. Their sensitivity against different antibiotics has been tested by culturing them with different antibiotic stabs for 72 h. Finally, Augmentin® was found as the most effective and suitable antibiotic which not only controlled the bacteria within 2–3 subcultures but also supported the regeneration system and growth of the regenerated shoots and such cultures have been grown for a long-term of over 2 years without any contamination.     

Thidiazuron: an efficient plant growth regulator for enhancing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant (4.1) were obtained on medium containing 2 mg l^?1 TDZ. Leaf explants inoculated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring ß-glucuronidase (uidA) and hygromycin resistance genes developed putative transformant shoots. The highest frequency of shoot regeneration (22.5%) and mean number of transformant shoots per explant (2.4) were obtained on a selection medium consisting of the above described regeneration medium and containing 25 mg l^?1 hygromycin as the selection agent. Approximately 95% of putative transformant shoots expressed the uidA gene following histochemical ß-glucuronidase (GUS) assay. These were confirmed to be transgenic by PCR analysis and Southern blot hybridization.     

In vitro propagation,ex vitro rooting and leaf micromorphology of <Emphasis Type="Italic">Bauhinia racemosa</Emphasis> Lam.: a leguminous tree with medicinal values

A micropropagation system for Bauhinia racemosa Lam. was developed involving axillary shoot proliferation and ex vitro rooting using nodal explants obtained from mature tree. MS medium with 3.0 mg l^?1 BA (6-benzyladenine) was optimum for shoot bud induction. For shoot multiplication, mother explants were transferred repeatedly on medium containing low concentration of BA (0.75 mg l^?1). Number of shoots was increased up to two passages and decreased thereafter. Shoot multiplication was further enhanced on MS medium containing 0.25 mg l^?1 each of BA and Kin (Kinetin) with 0.1 mg l^?1 of NAA (α-naphthalene acetic acid). Addition of 0.004 mg l^?1 TDZ (thidiazuron) increased the rate of shoot multiplication and 21.81 ± 1.26 shoots per culture vessel were obtained. In vitro regenerated shoots were rooted under ex vitro conditions treated with 400 mg l^?1 IBA (indole-3-butyric acid) for 7 min on sterile soilrite. After successful hardening in greenhouse, ex vitro rooted plants were transferred to the field conditions with ≈85% of survival rate. Micromorphological changes were observed on leaf surface i.e. development of vein density and trichomes and stomatal appearance, when plants were subjected to environmental conditions. This is the first report on in vitro regeneration of B. racemosa from mature tree.     

Agrobacterium-mediated transformation of Eruca sativa

An Agrobacterium tumefaciens—mediated transformation system was developed for Eruca sativa (eruca). Hypocotyl explants were co-cultivated with bacterial cells carrying a plasmid harboring a uidA:nptII fusion gene along a phosphinothricin acetyl transferase (PAT) gene cassette, for a period of 2 days. These were grown on a high cytokinin/auxin medium containing 5.0 mg l^?1 6-benzyladenine (BA), 1.0 mg l^?1 indole-3-acetic acid (IAA), and 0.1 mg l^?1 α-naphthaleneacetic acid (NAA). Explants were then transferred to a lower cytokinin/auxin medium containing 2.0 mg l^?1 BA and 0.1 mg l^?1 NAA along with 5.0 mg l^?1 silver nitrate and 300 mg l^?1 Timentin®. Upon transfer to a selection medium containing either 20 mg l^?1 kanamycin or 2 mg l^?1 L-phosphinothricin (L-ppt), shoot regenerants were observed. Expression of the transgenes in putative transformants was confirmed using a histochemical GUS assay. Presence of the PAT transgene in GUS-positive T0 plants was confirmed by Southern blot analysis. Moreover, spot tests of T1 seedlings were conducted using the L-ppt herbicide. A transformation frequency of 1.1% was obtained with more than 60% of transgenic lines containing single copies of the transgenes.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill.) somatic embryos with fluorescent marker genes and optimization of transgenic plant recovery

Avocado globular somatic embryos were transformed with three binary vectors, pK7FNF2, pK7RNR2 and pK7S*NF2, harboring the marker genes gfp, DsRed and a gfp-gus fusion gene, respectively. GFP and DsRed fluorescence was detected in embryogenic lines growing in selection medium 2 months after Agrobacterium inoculation. The fluorescence signal was maintained thereafter in transgenic calli, as well as in mature somatic embryos. Red fluorescence in pK7RNR2 transgenic lines was higher and more easily observable than GFP fluorescence. Furthermore, calli transformed with pK7S*NF2, harboring gfp-gus, showed higher level of fluorescence than those transformed with pK7FNF2, containing two gfp. To improve plant recovery, maturated transgenic embryos that failed to germinate or showed an underdeveloped shoot were cultured for 4 weeks in a medium with 1 mg l^?1 TDZ and 1 mg l^?1 BA after partial removal of cotyledons. A 50% of embryos developed one or several shoots on the cut surface. These embryos were cultured for 4 additional weeks in a medium with 1 mg l^?1 BA for shoot elongation and then, shoots were grafted in vitro onto seedling rootstocks. Culture of micrografts in solid MS medium supplemented with 1 mg l^?1 BA allowed a 60–80% success rate. Young leaves from transgenic plants showed GFP or DsRed fluorescence located in the nucleus. The results obtained indicate that fluorescent marker genes, especially DsRed, could be useful for early selection of transgenic material and optimization of the transformation parameters in avocado. Furthermore, the protocol established allowed the successful recovery of transgenic plants, one of the main limiting steps in avocado transformation.     

Transgenic <Emphasis Type="Italic">Eustoma grandiflorum</Emphasis> expressing the <Emphasis Type="Italic">bar</Emphasis> gene are resistant to the herbicide Basta<Superscript>®</Superscript>

Lisianthus (Eustoma grandiflorum) is a cut or ornamental flower that is popular all over the world. This ornamental crop, however, lacks an effective weed control method due to its susceptibility to herbicide. In this study, transgenic plants of a lisianthus cultivar were produced using Agrobacterium-mediated delivery of the plasmid pCAMBIA3300, which carried the bialaphos resistance (bar) gene under driven by the CaMV 35S promoter. The transgenic calli were derived from wounded edges of the leaves grown on a shoot regeneration medium containing 100 mg l^?1 cefotaxime and 2 mg l^?1 glufosinate ammonium for 4 weeks. The callus that was detached from the wounded edge of the leaf was transferred to the shoot regeneration medium with 100 mg l^?1 cefotaxime and 5 mg l^?1 glufosinate ammonium for 4 weeks for shoot regeneration. The bar gene integration and expression in the transgenic plants were confirmed by Southern and Northern blot analyses, respectively. Subsequently, the transgenic lines were assessed in vitro and under greenhouse conditions for their resistance to the commercial herbicide Basta^®, which contains glufosinate ammonium as the active component. Six transgenic lines showed high percentages (67–80%) of survival in vitro under the selection condition with glufosinate ammonium (up to 216 mg l^?1). Under greenhouse conditions, the plants from these six lines remained healthy and exhibited a normal phenotype after spraying with glufosinate ammonium (up to 1,350 mg l^?1). This is the first paper to provide a detailed survey of transgenic lisianthus expressing the bar gene and exhibiting herbicide-resistance under greenhouse conditions.     

Biomass and terpenoids produced by mutant strains of <Emphasis Type="Italic">Arthrospira</Emphasis> under low temperature and light conditions

The filamentous Cyanobacterium Arthrospira is commercially produced and is a functional, high-value, health food. We identified 5 low temperature and low light intensity tolerant strains of Arthrospira sp. (GMPA1, GMPA7, GMPB1, GMPC1, and GMPC3) using ethyl methanesulfonate mutagenesis and low temperature screening. The 5 Arthrospira strains grew rapidly below 14?°C, 43.75 μmol photons m^?2 s^?1 and performed breed conservation at 2.5?°C, 8.75 μmol photons m^?2 s^?1. We used morphological identification and molecular genetic analysis to identify GMPA1, GMPA7, GMPB1 and GMPC1 as Arthrospira platensis, while GMPC3 was identified as Arthrospira maxima. Growth at different culture temperatures was determined at regular intervals using dry biomass. At 16?°C and 43.75 μmol photons m^?2 s^?1, the maximum dry biomass production and the mean dry biomass productivity of GMPA1, GMPB1, and GMPC1 were 2057?±?80 mg l^?1, 68.7?±?2.5 mg l^?1 day^?1, 1839?±?44 mg l^?1, 60.6?±?1.8 mg l^?1 day^?1, and 2113?±?64 mg l^?1, 77.7?±?2.5 mg l^?1 day^?1 respectively. GMPB1 was chosen for additional low temperature tolerance studies and growth temperature preference. In winter, GMPB1 grew well at mean temperatures <10?°C, achieving 3258 mg dry biomass from a starting 68 mg. In summer, GMPB1 grew rapidly at mean temperatures more than 28?°C, achieving 1140 mg l^?1 dry biomass from a starting 240 mg. Phytonutrient analysis of GMPB1 showed high levels of C-phycocyanin and carotenoids. Arthrospira metabolism relates to terpenoids, and the methyl-d-erythritol 4-phosphate pathway is the only terpenoid biosynthetic pathway in Cyanobacteria. The 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) gene from GMPB1 was cloned and phylogenetic analysis showed that GMPB1 is closest to the Cyanobacterium Oscillatoria nigro-viridis PCC711. Low temperature tolerant Arthrospira strains could broaden the areas suitable for cultivation, extend the seasonal cultivation time, and lower production costs.     

Characterization of alcohol dehydrogenase from <Emphasis Type="Italic">Kangiella koreensis</Emphasis> and its application to production of all-<Emphasis Type="Italic">trans</Emphasis>-retinol

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min^?1 mg^?1, a K _m of 1.8 μM, and a k _cat of 1.7 min^?1 for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD ⁺ as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l^?1, and 2,200 mg all-trans-retinal l^?1 in the presence of 5 % (v/v) methanol, 1 % (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l^?1 after 3 h, with a conversion yield of 27.3 % (w/w) and a productivity of 200 mg l^?1 h^?1. This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.     

Micropropagation, <Emphasis Type="Italic">in vitro</Emphasis> flowering,and tuberization in <Emphasis Type="Italic">Brachystelma glabrum</Emphasis> Hook.f., an endemic species

Brachystelma glabrum Hook.f. is an endemic plant species of Eastern Ghats, India. In this study, efficient protocols for in vitro micropropagation, flowering, and tuberization of this plant were developed. Sterilized shoot tip and nodal explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators (PGRs) and additives for shoot induction and multiplication. Both shoot tip and nodal explants showed the best response (90 and 100%, respectively) on MS medium supplemented with thidiazuron (TDZ) at 1.0 mg L^?1. The microshoots multiplied best on MS + TDZ (1.0 mg L^?1) in combination with α-naphthaleneacetic acid (NAA) at 0.5 mg L^?1 and coconut water (CW) at 25%. The highest number of in vitro flowers (4.0 flowers per microshoot) was observed on MS medium supplemented with a combination of N6-benzyladenine (BA) and indole-3-butyric acid (IBA), each at 1.5 mg L^?1. In vitro-derived shoots produced aerial tubers on MS + TDZ (2.0 mg L^?1) + IBA (0.5 mg L^?1) and basal tubers on MS + TDZ at 2.0 mg L^?1. In vitro shoots were best rooted on half-strength (½) MS + NAA at 0.5 mg L^?1. The rooted plantlets were successfully acclimatized in pots with 70% survival after a hardening period of 1 mo. This protocol provides an effective method for the conservation of this endemic plant species.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration,production, and storage of artificial seeds in <Emphasis Type="Italic">Ceropegia barnesii</Emphasis>, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L^?1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L^?1 benzyl adenine and 1 mg L^?1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and shoot regeneration in elite breeding lines of western shipper cantaloupe and honeydew melons (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

A reliable Agrobacterium-mediated transformation and shoot regeneration protocol was developed for breeding lines of commercially important western-shipper cantaloupe and honeydew melons, ‘F39’ and ‘150’, respectively. Different media were tested to select a shoot regeneration system for each of three elite breeding lines ‘F39’, ‘141’ and ‘TMS’. Murashige &; Skoog (MS) basal medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.26 mg l^?1 abscisic acid (ABA) and 0.8 mg l^?1 indole-3-acetic acid (IAA) was used for shoot regeneration from cotyledonary explants in ‘F39’ and ‘150’. Kanamycin sensitivity as well as Timentin^? and Clavamox^® were evaluated using wild-type ‘F39’ and ‘150’ cotyledons. Kanamycin concentrations of 200 and 150 mg l^?1 were chosen as the threshold levels for ‘F39’ and ‘150’, respectively. No significant differences were found between Timentin^? and Clavamox^® in ‘F39’; however, Clavamox^® reduced the incidence of vitrification and increased the frequency of shoot elongation in ‘150’. A. tumefaciens strain EHA105, harboring pCNL56 carrying neomycin phosphotransferase II (nptII) and gusA reporter genes, was selected to establish a transformation protocol for ‘F39’ and ‘150’. Putative transformants were evaluated using β-glucuronidase (GUS) histochemical assay, polymerase chain reaction (PCR) and Southern blot analyses. Based on these parameters, the transformation efficiency for cantaloupe ‘F39’ was 0.3% and that for honeydew ‘150’ was 0.5%.     

Quercetin and silver nitrate modulate organogenesis in <Emphasis Type="Italic">Carissa carandas</Emphasis> (L.)

An in vitro organogenesis protocol for Carissa carandas L. was developed using an auxin transport inhibitor (quercetin) and silver nitrate (AgNO₃), an inhibitor of ethylene action, in association with cytokinins in the culture medium. This protocol produced the maximum number of shoots from aseptic seedling-derived shoot apex explants of C. carandas. The highest rate of shoot multiplication was recorded on MS medium containing 2.0 mg L^?1 6-benzylaminopurine; 0.5 mg L^?1 kinetin, and 0.75 mg L^?1 quercetin at after 4 wk of culture. Similar results were obtained when MS medium fortified with 2.0 mg L^?1 BAP, 0.5 mg L^?1 kinetin, and 1.5 mg L^?1 AgNO₃ was used. However, successful rooting was achieved on quarter strength MS medium with 0.5 mg L^?1 indole-3-acetic acid. In this study, an inhibitor of auxin transport and ethylene action maximized shoot multiplication in medium fortified with cytokinins. The established rapid micropropagation method could be used to conserve elite genotypes of C. carandas.     

Combinatorial analysis of enzymatic bottlenecks of <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine pathway by <Emphasis Type="Italic">p</Emphasis>-coumaric acid production in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Objective

To identify new enzymatic bottlenecks of l-tyrosine pathway for further improving the production of l-tyrosine and its derivatives.

Result

When ARO4 and ARO7 were deregulated by their feedback resistant derivatives in the host strains, the ARO2 and TYR1 genes, coding for chorismate synthase and prephenate dehydrogenase were further identified as new important rate-limiting steps. The yield of p-coumaric acid in the feedback-resistant strain overexpressing ARO2 or TYR1, was significantly increased from 6.4 to 16.2 and 15.3 mg l^?1, respectively. Subsequently, we improved the strain by combinatorial engineering of pathway genes increasing the yield of p-coumaric acid by 12.5-fold (from 1.7 to 21.3 mg l^?1) compared with the wild-type strain. Batch cultivations revealed that p-coumaric acid production was correlated with cell growth, and the formation of by-product acetate of the best producer NK-M6 increased to 31.1 mM whereas only 19.1 mM acetate was accumulated by the wild-type strain.

Conclusion

Combinatorial metabolic engineering provides a new strategy for further improvement of l-tyrosine or other metabolic biosynthesis pathways in S. cerevisiae.

     

High responsiveness in <Emphasis Type="Italic">de novo</Emphasis> shoot organogenesis induction of <Emphasis Type="Italic">Passiflora cristalina</Emphasis> (Passifloraceae), a wild Amazonian passion fruit species

The aim of the present study was to establish a regeneration system via de novo organogenesis from different types of non-meristematic explants of Passiflora cristalina. Leaf, hypocotyl, root segments, cotyledons, and endosperm of P. cristalina seeds were inoculated in Murashige and Skoog (MS)-basal medium, supplemented with different concentrations of 6-Benzyladenine (BA), Thidiazuron (TDZ), or Kinetin (KIN). BA was found to be the most efficient cytokinin in induction of de novo organogenesis from most the explants used in the study. The highest frequencies of adventitious bud formation in the hypocotyl and cotyledon explants were observed in medium supplemented with 1.0 mg L^?1 BA. For leaf and endosperm segments, the best concentration was 2.0 mg L^?1 BA; while for root segments, the highest mean values were observed with 1.0 mg L^?1 KIN. The different morphogenetic responses obtained from each explant source were characterized using light microscopy. P. cristalina revealed a remarkable organogenic potential, with superior production of adventitious shoots compared with the other Passiflora species evaluated elsewhere. These results will be helpful to establish a reproducible and reliable micropropagation protocol, as well as to implement conservationist and biotechnological-based genetic breeding strategies for this wild Passiflora species.     

Efficient plant regeneration of the endangered medicinal orchid, <Emphasis Type="Italic">Coelogyne cristata</Emphasis> using protocorm-like bodies

An efficient method of Coelogyne cristata mass propagation was developed using segment of protocorm-like bodies (PLBs) (3 mm² in size). It was observed that ½ MS medium showed to be more effective to induce shoots through PLBs segment. The explants when cultured on ½ MS media containing TDZ and CP showed relatively superior effect on shoot regeneration as compared to the media containing TDZ alone or in combination with BP. Addition of BP and CP to the medium containing NAA and BA combinations proved distinctly better for shoot multiplication than that of the medium with NAA and BA combinations alone. The highest percentage of explants producing shoots, with a maximum average of 8.1 per explant, was induced on the medium supplemented with 1.0 mg l^?1 NAA and 0.5 mg l^?1 BA with CP. Shoots produced an average of 15 roots per explant on ½ MS medium supplemented with 2.0 mg l^?1 IBA and BP. The 4 cm height plantlets with well-developed roots were successfully acclimatized. The results suggest that CP and BP can be used effectively to initiate shooting and rooting of Coelogyne cristata. Ploidy analysis of regenerated plants using flow cytometry revealed the same ploidy level (diploid). This efficient and reliable protocol could be useful for mass multiplication and germplasm conservation of the wild medicinal orchid.