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.     

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.     

Plant regeneration of the arsenic hyperaccumulator <Emphasis Type="Italic">Pteris vittata</Emphasis> L<Emphasis Type="Italic">.</Emphasis> from spores and identification of its tolerance and accumulation of arsenic and copper

An in vitro plant regeneration system was established from the spores of Pteris vittata and identification of its tolerance, and accumulation of gametophytes and callous, to arsenic (As) and copper (Cu) was investigated. The highest frequency (100%) of callus formation was achieved from gametophyte explants treated with 0.5 mg l^?1 6-benzylaminopurine (6-BA) + 0.5 mg l^?1 gibberellin acid (GA). Furthermore, sporophytes were differentiated from the callus tissue derived from gametophyte explants on MS medium supplemented with 0.5 mg l^?1 6-BA, 0.5–1.0 mg l^?1 GA and additional 300 mg l^?1 lactalbumin hydrolysate (LH) for 4 weeks. The optimum combination of ½ MS + 1.0 mg l^?1 GA + 0.5 mg l^?1 6-BA + 300 mg l^?1 LH promoted sporophyte formation on 75 ± 10% of the callus. Every callus derived from gametophyte explants could achieve 3–4 sporophytes. The in vitro growth of gametophyte and callus was accelerated in the medium containing Na₃AsO₄ lower than 0.5 mM, but this growth was inhibited with 2 mM Na₃AsO₄. And with the increase of Na₃AsO₄ in the culture medium from 0 to 2 mM, the As accumulation in gametophytes and callus increased and achieved a level of 763.3 and 315.4 mg kg^?1, respectively. Gametophytes and calluses transplanted to culture medium, supplemented with different concentrations of CuSO₄, are similar to those in Na₃AsO₄, and the Cu accumulation in gametophytes could achieve 7,940 mg kg^?1 when gametophytes were subcultured in medium containing 3 mM CuSO₄. These results suggested that the high efficiency propagation system could be a useful and rapid means to identify other heavy metal tolerance and accumulation. Further, the regeneration ability of callus made it possible for genetic transformation of this fern.     

Optimizing micropropagation of drought resistant <Emphasis Type="Italic">Pyrus boissieriana</Emphasis> Buhse

The present study concentrated on introducing a micropropagation protocol for a drought resistant genotype from Pyrus boissieriana, which is the second most naturally widespread pear species in Iran with proper physiological and medicinal properties. Proliferating microshoot cultures were obtained by placing nodal segments on MS medium supplemented with BAP and IBA or NAA. The highest number of shoots (27 shoots per explant) were obtained with 1.5 mg l^?1 BAP and 0.05 mg l^?1 IBA, but this combination did not produce shoots of desirable length (>1.7 cm). Combination of 1.75 mg l^?1 BAP and 0.07 mg l^?1 IBA was the best for the shoot multiplication in P. boissieriana with a sufficient number of shoot production (22.33 shoots per explant) and relatively more appropriate shoot length. The larger and greenish leaves were obtained when PG was added to the best multiplication treatment. Microshoot elongation was carried out in 1/2 and 1/4 MS medium containing 50–100 mg l^?1 PG with different concentrations of IBA or NAA at intervals of 30–60 days. Significant increase in shoot length was detected after 45–60 days of culture in the presence of PG. The highest shoot length (8 cm) was recorded on 1/2 MS medium supplemented with 0.5 mg l^?1 IBA and 100 mg l^?1 PG. GA₃ negatively affected number and length of shoots and generally caused generation of red leaves. The highest percentage of root induction (100%) and root length (9 cm) were obtained on 1/6 strength MS medium supplemented with 0.005 mg l^?1 IBA. All plantlets were hardened when transferred to ex vitro conditions through a period of 25–30 days. The results suggest axillary shoot proliferation of P. boissieriana could successfully be employed for propagation of candidate drought resistant seedling.     

<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.     

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">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.     

The synergistic effects of sucrose and plant growth regulators on morphogenesis and evaluation of antioxidant activities in regenerated tissues of <Emphasis Type="Italic">Caralluma tuberculata</Emphasis>

Caralluma tuberculata (C. tuberculata) is a very important medicinal plant with a range of anti-diabetic and weight reduction properties. This high-valued medicinal plant is nowadays considered as endangered due to its unsustainable elimination from wild habitats. There is lack of research efforts on its propagation to overcome escalating demand. In this research study, an effort has been made to optimize protocol for large-scale mass propagation and production of natural antioxidants. Highest callogenic response (87.2 %) was observed from shoot tip explants on Murashige and Skoog (MS) medium containing 30 g l^?1 sucrose and combination of 2, 4-D (2.0 mg l^?1) and BA (1.0 mg l^?1). During shoot morphogenesis, 50 g l^?1 sucrose along with BA (2.0 mg l^?1) and GA₃ (1.0 mg l^?1) enhanced shoot regeneration (91.3 %), mean shoot length (2.6 cm) and shoots per explant (24.5) as compared to control. The combination of IBA and IAA (2.0 mg l^?1) was found optimum for root induction (74.98 %), mean root length (4.1 cm) and roots per shoot (6.9) as compared to control. The plantlets were successfully acclimatized in plastic cups and various tissues were investigated for accumulation of antioxidant secondary metabolites including phenolics, flavonoids, stress enzymes and antioxidant activities. The superoxide dismutase enzyme was higher in shoots; protein content was higher in callus cultures; phenolics, DPPH and protease activity were higher in plantlets, while flavonoids, peroxidase, reducing power and total antioxidant activities were higher in wild plants. This simple protocol is very useful for commercial production of consistent plantlets and metabolites of interest.     

In vitro tetraploidization for the augmentation of wedelolactone in <Emphasis Type="Italic">Sphagneticola calendulacea</Emphasis> (L.) Pruski

A practical and reliable method for in vitro tetraploidization of Sphagneticola calendulacea (L.) Pruski [synonym Wedelia chinensis (Osbeck) Merrill] has been established to enhance the production of wedelolactone. Shoot tip and nodal explants from in vitro-grown culture (2n?=?50) were exposed to the antimitotic chemical, i.e., colchicine, at various concentrations (0, 0.025, 0.05, 0.1, 0.3, and 0.5%; w/v) for 12, 24, 36, 48, and 60 h. The treated explants were then incubated and proliferated on Murashige and Skoog (MS) medium fortified with 0.2 mg l^?1 thidiazuron and 0.05 mg l^?1 naphthalene acetic acid, followed by root induction in 1.0 mg l^?1 indole-3 acetic acid enriched ½MS medium. Treatment of shoot tips with 0.05% colchicine for 24 h supported the maximum rate of survival (63.33%) of explants as well as tetraploid induction (42.93%). Morphological, stomatal, and cytological characteristics along with the secondary metabolite content of the in vitro tetraploids were compared to that of diploids. The recovered tetraploid plants possessed superior plant height, stem diameter, leaf size, root number, and increased length and width of stomata but decreased stomatal frequency. The tetraploid plants demonstrated twice the chromosome number (2n?=?4x?=?100) than the diploids as confirmed through cytology, spectrophotometry and flow cytometry. High-performance thin-layer chromatography showed a significant enhancement in the wedelolactone content of tetraploid plants (541.48 µg g^?1 of dried sample) in comparison to diploid plants (325.43 µg g^?1 of dried sample), signifying the prospective of this technique for the trade value improvement.     

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.     

Isolation,expression, and evolution analysis of the type 2C protein phosphatase gene <Emphasis Type="Italic">BcABI1</Emphasis> involved in abiotic and biotic stress in <Emphasis Type="Italic">Brassica campestris</Emphasis> ssp. <Emphasis Type="Italic">chinensis</Emphasis>

Dense plant cultivation is an efficient approach to utilize the maximum inputs for increasing maize production. However, dense plant populations may prone to lodging as it results in increased plant height and reduced culm diameter; therefore, we hypothesized that weaker stems may be responsible for maize lodging. In this study, we examined the regulatory effects of paclobutrazol under two commonly used application methods (seed-soaking and seed-dressing). Seed-soaking with paclobutrazol at the rate of 0 (CK1), 200 (S1), 300 (S2), and 400 (S3) mg L^?1, while seed-dressing at the rate of 0 (CK2), 1.5 (D1), 2.5 (D2), and 3.5 (D3) g kg^?1 were used. Results showed that paclobutrazol improved the culm physical strength by increasing the rind penetration strength, stalk breaking strength, culm diameter, wall thickness, and dry weight per unit length of basal third internode, compared to control plants. Moreover, paclobutrazol reduced the internode length, plant height, ear height, center of gravity height and lodging rate in both growing seasons. In addition, more lignin was accumulated in the basal internode and the activities of phenylalanine ammonia-lyase (PAL), peroxidase (POD), cinnamyl alcohol dehydrogenase (CAD) and 4-coumarate: CoA ligase (4CL) increased with paclobutrazol, and their maximum values were observed in the S2 and D3 treatments, resulting in strong lodging resistance. Lignin content was positively and significantly correlated with the rind penetration strength, breaking strength of internode, and activities of PAL, 4CL, POD, and CAD, while significantly and negatively correlated with lodging percentage. The present findings suggested that 300 mg L^?1 and 3.5 g kg^?1 of paclobutrazol may efficiently be utilized to minimize the risk of lodging, not only by manipulating plant height but also by enhancing culm physical strength and lignin accumulation in basal internodes.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Exploring <Emphasis Type="Italic">in vitro</Emphasis> germplasm conservation options for sugarcane (<Emphasis Type="Italic">Saccharum</Emphasis> spp. hybrids) in South Africa

In vitro plantlets of sugarcane cultivar NCo310 were maintained in slow growth conditions at both 18 and 24°C and on four semi-solid media: SG1—Murashige and Skoog (MS) salts and vitamins with 20 g L^?1 sucrose, SG2—½ MS with 10 g L^?1 sucrose, SG3—MS with 20 g L^?1 sucrose and 1 mg L^?1 abscisic acid (ABA), and SG4—½ MS with 10 g L^?1 sucrose and 1 mg L^?1 ABA. After 8, 12, 24, 36, and 48 mo shoot multiplication rates were recorded, shoots were removed from storage and subcultured every 2 wk on SG1 with 0.015 mg L^?1 kinetin and 0.1 mg L^?1 benzyl aminopurine for 2 mo. At 18°C, all media supported storage for 48 mo with subculturing every 12 mo. Shoot multiplication post-retrieval was significantly higher on the SG2 medium compared with the non-stored control (362 ± 84 and 126 ± 26 shoots per recovered shoot after 2 mo, respectively). In addition, shoots could be maintained for 48 mo on SG2 medium with one subculture without compromising post-storage multiplication ability. At 24°C, storage on all four media supported recovery and multiplication of shoots for 8 mo and only SG2 medium facilitated survival for 12 mo. There was no advantage to incorporating ABA into the storage media, regardless of the temperature and storage time. Cryopreservation of cultivar NCo376 in vitro-derived shoot meristems using the V-cryo-plate method demonstrated that the sucrose concentration in the loading solution (0.8–1.8 M) had no significant effect on survival of the meristems, which ranged from 41.7 ± 4.8 to 69.4 ± 10%.     

<Emphasis Type="Italic">In vitro</Emphasis> sucrose concentration influences microtuber production and diosgenin content in white yam (<Emphasis Type="Italic">Dioscorea rotundata</Emphasis> Poir)

Dioscorea spp. is an important food crop in many countries and the source of the phytochemical diosgenin. Efficient microtuber production could provide source materials for farm-planting stock, for food markets, and for the production of high-diosgenin-producing cultivars. The first step in this study was optimizing the plant growth regulators for plantlet production, followed by a study of the effects of sucrose concentration on microtuber induction and diosgenin production. Significantly, more shoots (3.5) were produced at 4.65 μM (1 mg L^?1) kinetin (KIN), longer shoots (4.1 cm) were obtained at 2.46 μM (0.5 mg L^?1) indole-3-butyric acid (IBA), and root number (3.9) was significantly higher at 5.38 μM (1 mg L^?1) naphthalene acetic acid (NAA) than in other treatments. Increased sucrose concentrations in the optimized growth medium with 4.65 μM KIN and 5.38 μM NAA had significant effects on microtuber production (p < 0.01) and diosgenin content (p < 0.05). The most microtubers (6.2) were obtained with 100 g L^?1 sucrose, while those on 80 g L^?1 sucrose were the heaviest (0.7 g) and longest (7.4 mm). Microtubers formed in medium with 80 g L^?1 sucrose had significantly higher diosgenin content (3.64% [w/w]) than those in other sucrose treatments (< 2%) and was similar to that of field-grown parent tubers (3.79%). This result indicates an important role for sucrose in both microtuber growth and diosgenin production. Medium containing 4.65 μM KIN and 5.38 μM NAA is recommended for plantlet production, and medium containing 80 g L^?1 sucrose is recommended for microtuber and diosgenin production.     

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.     

<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%.     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

Raw starch conversion by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> amylases

Background

Starch is one of the most abundant organic polysaccharides available for the production of bio-ethanol as an alternative transport fuel. Cost-effective utilisation of starch requires consolidated bioprocessing (CBP) where a single microorganism can produce the enzymes required for hydrolysis of starch, and also convert the glucose monomers to ethanol.

Results

The Aspergillus tubingensis T8.4 α-amylase (amyA) and glucoamylase (glaA) genes were cloned and expressed in the laboratory strain Saccharomyces cerevisiae Y294 and the semi-industrial strain, S. cerevisiae Mnuα1. The recombinant AmyA and GlaA displayed protein sizes of 110–150 kDa and 90 kDa, respectively, suggesting significant glycosylation in S. cerevisiae. The Mnuα1[AmyA-GlaA] and Y294[AmyA-GlaA] strains were able to utilise 20 g l^-1 raw corn starch as sole carbohydrate source, with ethanol titers of 9.03 and 6.67 g l^-1 (0.038 and 0.028 g l^-1 h^-1), respectively, after 10 days. With a substrate load of 200 g l^-1 raw corn starch, Mnuα1[AmyA-GlaA] yielded 70.07 g l^-1 ethanol (0.58 g l^-1 h^-1) after 120 h of fermentation, whereas Y294[AmyA-GlaA] was less efficient at 43.33 g l^-1 ethanol (0.36 g l^-1 h^-1).

Conclusions

In a semi-industrial amylolytic S. cerevisiae strain expressing the A. tubingensis α-amylase and glucoamylase genes, 200 g l^-1 raw starch was completely hydrolysed (saccharified) in 120 hours with 74% converted to released sugars plus fermentation products and the remainder presumably to biomass. The single-step conversion of raw starch represents significant progress towards the realisation of CBP without the need for any heat pretreatment. Furthermore, the amylases were produced and secreted by the host strain, thus circumventing the need for exogenous amylases.

     

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.     

Micropropagation of <Emphasis Type="Italic">Asparagus macrorrhizus</Emphasis>, a Spanish endemic species in extreme extinction risk

Asparagus macrorrhizus: is a new species, which has been recently described. It is limited to the area surrounding the “Mar Menor” lagoon, in Murcia (Spain), and is the only “Critically Endangered” species of the genus Asparagus. Despite being protected, the number of plants has decreased in the last years due to the urbanization of its natural habitat. This species is a valuable genetic resource for asparagus breeding because of its special characteristics. So, the development of a micropropagation protocol is crucial to its conservation and use in breeding programs. The micropropagation protocol from asparagus rhizome buds previously developed by our research group has been adapted for A. macrorrhizus. Rhizome buds of A. macrorrhizus were extracted, disinfected, and then cultured on Asparagus Rhizome Bud Medium (ARBM) consisting of MS medium supplemented with 0.3 mg l^??1 NAA, 0.1 mg l^??1 KIN, 2 mg l^??1 ancymidol and 6% sucrose. A percentage of 69.7?±?8.0% of the rhizome buds developed shoots, but only 17.4?±?7.9% of them rooted. To increase this low rooting rate, the shoots were cultured on Macrorrhizus Rooting Media (MRM) supplemented with three different concentrations of IBA. The highest rooting rate (55.0?±?7.9%) was reached when shoots were incubated in MRM-2 consisting of MS medium supplemented with 2 mg l^??1 IBA and 4% sucrose. The acclimatization rate of the micropropagated plantlets was 90%. The method developed in this study allows the micropropagation of A. macrorrhizus, offering a new option to preserve this almost extinct species.