Production of sapogenins (stigmasterol and hecogenin) from genetically transformed hairy root cultures of <Emphasis Type="Italic">Chlorophytum borivilianum</Emphasis> (Safed musli)

Chlorophytum borivilianum belonging to the family Liliaceae, is distributed in the pantropical regions of India and South Africa. The sapogenins (stigmasterol and hecogenin) of C. borivilianum are well known for their appetizing and aphrodisiac properties. The present study involves enhancing the sapogenin content in C. borivilianum by genetic transformations with Agrobacterium rhizogenes strains (MTCC 2364 and 532, PRT Gus). A maximum transformation frequency of 98% was obtained with Agrobacterium rhizogenes MTCC 2364 strain with rhizome explants after a co-cultivation period of 48 h. Two potential rhizoclones (2364a and 2364b) were selected for the production of stigmasterol and hecogenin. The maximum production of stigmasterol (83.952?±?0.01 mg/g) was seen in 2364b rhizoclone, whereas, the highest accumulation of hecogenin (81.52?±?0.02 mg/g) was observed in 2364a rhizoclone. The C. borivilianum hairy root cultures obtained in this study provide a continuous and sustainable production of stigmasterol and hecogenin on a commercial scale.     

Impact of Proteolytic <Emphasis Type="Italic">Lactobacillus helveticus</Emphasis> MTCC5463 on Production of Bioactive Peptides Derived from Honey Based Fermented Milk

In this study, Lactobacillus helveticus MTCC5463 was evaluated for its proteolytic activity and production of bioactive peptides during fermentation of honey supplemented milk under specified growth conditions. Generally, lactic acid bacteria have a strong proteolytic system. However, L. helveticus MTCC5463 showed maximum proteolytic activity at 4 % level of honey supplementation compared to 6 % and control. Similarly, water soluble extract derived from fermented honey based milks exhibited different level of bioactive peptides productions during fermentation. L. helveticus MTCC5463 showed maximum peptides production at 4 % level of honey supplementation compared to control during HPLC analysis and LC–MS analysis.     

Optimization of a <Emphasis Type="Italic">Bacopa monnieri</Emphasis>-based genetic transformation model for testing the expression efficiency of pathway gene constructs of medicinal crops

A fast regenerating Agrobacterium tumefaciens-mediated transformation protocol for Bacopa monnieri (L.) Wettst. was developed as a model system for heterologous expression of terpenoid indole alkaloid pathway genes from Catharanthus roseus (L.) G. Don. The direct regeneration of shoots from leaf explants co-cultured with A. tumefaciens resulted in the integration of a tryptophan decarboxylase (tdc) and strictosidine synthase (str) cassette (<hpt-<Tdc2-<Str-gus>) in the regenerated progeny. The highest transformation efficiency (83.88%) was achieved when leaf explants were infected on the adaxial laminar surface by manual pricking with 48- to 72-h-old suspensions (OD₆₀₀ = 0.5–0.6) of A. tumefaciens strain LBA1119 (carrying the binary vector pMOG22). The heterologous expression of tryptophan decarboxylase and strictosidine synthase genes that are otherwise not present in B. monnieri plants was confirmed through semi-quantitative PCR and metabolite quantification assays. The entire protocol duration from co-cultivation through regeneration of transgenic plants to their establishment in the glass house took 40–45 d. The developed B. monnieri model can be used to test expression cassettes carrying genes for plant secondary metabolic pathway engineering, especially those genes that are expressed in differentiated cell, tissue, or organs.     

Evaluation of genetic and phenotypic consistency of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> MTCC 5856: a commercial probiotic strain

Commercial probiotics preparation containing Bacillus coagulans have been sold in the market for several decades. Due to its high intra-species genomic diversity, it is very likely that B. coagulans strain may alter in different ways over multiple years of production. Therefore, the present study focuses to evaluate the genetic consistency and probiotic potential of B. coagulans MTCC 5856. Phenotypic and genotypic techniques including biochemical profiling, 16S rRNA sequencing, GTG 5″, BOX PCR fingerprinting, and Multi-Locus-Sequence typing (MLST) were carried out to evaluate the identity and consistency of the B. coagulans MTCC 5856. Further, in vitro probiotic potential, safety and stability at ambient temperature conditions of B. coagulans MTCC 5856 were evaluated. All the samples were identified as B. coagulans by biochemical profiling and 16S rRNA sequencing. GTG 5″, BOX PCR fingerprints and MLST studies revealed that the same strain was present over 3 years of commercial production. B. coagulans MTCC 5856 showed resistance to gastric acid, bile salt and exhibited antimicrobial activity in in-vitro studies. Additionally, B. coagulans MTCC 5856 was found to be non-mutagenic, non-cytotoxic, negative for enterotoxin genes and stable at ambient temperature (25 ± 2 °C) for 36 months. The data of the study verified that the same strain of B. coagulans MTCC 5856 was present in commercial preparation over multiple years of production.     

Yeast-extract improved biosynthesis of lignans and neolignans in cell suspension cultures of <Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.

Lignans and neolignans are important biologically active ingredients (BAIs) biosynthesized by Linum usitatissimum. These BAIs have multi-dimensional effects against cancer, diabetes and cardio vascular diseases. In this study, yeast extract (YE) was employed as an elicitor to evaluate its effects on dynamics of biomass, BAIs and antioxidant activities in L. usitatissimum cell cultures. During preliminary experiments, flax cultures were grown on different concentrations of YE (0–1000 mg/L), and 200 mg/L YE was found to be optimum to enhance several biochemical parameters in these cell cultures. A two-fold increase in fresh (FW) and dry weight (DW) over the control was observed in cultures grown on MS medium supplemented with 200 mg/L YE. Similarly, total phenolic (TPC; 16 mg/g DW) and flavonoids content (TFC; 5.1 mg/g DW) were also positively affected by YE (200 mg/L). Stimulatory effects of YE on biosynthesis of lignans and neolignans was also noted. Thus, 200 mg/L of YE enhanced biosynthesis of secoisolariciresinol diglucoside (SDG; 3.36-fold or 10.1 mg/g DW), lariciresinol diglucoside (LDG; 1.3-fold or 11.0 mg/g DW) and dehydrodiconiferyl alcohol glucoside (DCG; 4.26-fold or 21.3 mg/g DW) in L. usitatissimum cell cultures with respect to controls. This elicitation strategy could be scaled up for production of commercially feasible levels of these precious metabolites by cell cultures of Linum.     

<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.     

Diversity of low-molecular weight organic acids synthesized by <Emphasis Type="Italic">Salix</Emphasis> growing in soils characterized by different Cu,Pb and Zn concentrations

The aim of the study was to evaluate the biosynthesis and exudation of 10 low-molecular weight organic acids (LMWOAs) into the rhizosphere with a simultaneous analysis of the acid contents in the roots and leaves of 9 Salix taxa growing on two experimental areas, differing in their concentrations of copper (Cu), lead (Pb) and zinc (Zn) in the soil (Area 1—low, Area 2—high concentration). The obtained results reveal a significant difference in the phytoextraction of the tested Salix taxa for the analysed metals in both areas. The highest contents of Cu, Pb and Zn were observed for all Salix collected from Area 2, especially in S. × smithiana roots (116 ± 8.76, 87.84 ± 7.30 and 203.42 ± 14.62 mg kg^?1 DW, respectively). The results obtained in Area 2 also revealed acidification of the rhizosphere and a higher concentration of acids, mainly oxalic, malic, malonic, acetic and citric acids. Contents of oxalic, malic, acetic and citric acids increased in the roots of Salix taxa from Area 2, while in the leaves formic and succinic acids were also present. S. × smithiana was the taxon with the highest concentration of acids in the rhizosphere and roots (73.48 ± 6.77 and 49.79 ± 2.65 μM 100 g^?1 DW, respectively), while in leaves a higher content was observed for S. alba and S. viminalis ‘PR’ taxa (78.12 ± 3.95 and 71.12 ± 3.75 μM 100 g^?1 DW, respectively).     

Enhancement of stilbene compounds and anti-inflammatory activity of methyl jasmonate and cyclodextrin elicited peanut hairy root culture

A highly stable and productive hairy root culture from peanut cultivar Tainan9 (T9-K599) was established using Agrobacterium rhizogenes strain K599 (NCPPB 2659)-mediated transformation. Valuable phenolic compounds with antioxidant activity and stilbene compounds were produced and secreted into the culture medium after elicitation with 100 µM methyl jasmonate (MeJA) and 6.87 mM cyclodextrin (CD). The antioxidant activity of the culture medium was increased to the highest Trolox equivalent antioxidant capacity (TEAC) value (28.30?±?2.70 mM Trolox/g DW) in the group treated with CD. The group co-treated with MeJA and CD exhibited the highest phenolic content, with a gallic acid equivalent (GAE) value of 10.80?±?1.00 µg gallic acid/g DW. The CuZn-SOD (CuZn superoxide dismutase) and APX (ascorbate peroxidase) antioxidant enzyme gene were up-regulated in the treatment with CD alone while the CuZn-SOD, GPX (glutathione peroxidase) and APX gene expression were down-regulated in the co-treatment with MeJA plus CD. The stilbene compounds resveratrol, trans-arachidin-1 and trans-arachidin-3 were detected by analysing the culture medium treated with CD alone and after co-treatment with MeJA and CD via HPLC. The LC-MS/MS results confirmed the presence of resveratrol, trans-arachidin-1, trans-arachidin-3, 4-Isopentadienyl-3,5,3′,4′-tetrahydroxystilbene (IPP), trans-3′-Isopentadienyl-3,5,4′-trihydroxystilbene (IPD) and arahypin-7. The results indicate that elicited peanut hairy roots can produce beneficial stilbene compounds that have antioxidant properties and anti-inflammatory activity. This peanut hairy root system could be applied as an experimental model to enhance the production of stilbene and other polyphenolic bioactive compounds.     

<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.     

Seasonal variations in harvest index and bacoside A contents amongst accessions of <Emphasis Type="Italic">Bacopa monnieri</Emphasis> (L.) Wettst. collected from wild populations

Bacoside A, a major active principle of Bacopa monnieri known for its cognitive effects is a mixture of saponins like bacoside A3, bacopaside II, isomer of bacopasaponin C and bacopasaponin C. Seasonal changes in biomass and bacoside A levels in fourteen accessions of B. monnieri were evaluated after maintaining these at a common site at Thapar University campus, Patiala (30°19′36.12″N and 76°24′1.08″E) for 1 year. Harvestable biomass and total bacoside A contents varied significantly between the accessions and also in a particular accession during different seasons of the year. The maximum dry weight of plant (biomass 1.64 g) and bacoside A levels (6.82 mg/plant) were recorded in accession BM1. Harvestable biomass was highest during summer in accessions BM1 and BM7 (FW 4.2 g/plant), whereas bacoside A levels were also highest during summer and in accession BM1 (6.82 mg/plant). The lowest bacoside A level (0.06 mg/plant) was recorded in accession BM14 during winter. Principal component analysis showed that samples of summer were positively correlated with both the components suggesting an appropriate time for the harvest.     

A rapid and efficient in vitro shoot regeneration protocol using cotyledons of London plane tree (<Emphasis Type="Italic">Platanus acerifolia</Emphasis> Willd.)

A rapid, prolific and reproducible protocol for in vitro shoot regeneration from mature cotyledons of Platanus acerifolia has been developed. The influences of different plant growth regulator (PGR) combinations and donor seedling ages on shoot regeneration were investigated. The results showed that the application of BA in conjunction with NAA was the most effective PGR combination for the induction of shoot regeneration. When cotyledon explants of 5-day-old seedlings were incubated on MS basal medium supplemented with 4.0 mg L^?1 BA and 0.2 mg L^?1 NAA, 67.6?±?4.9% of the cotyledon segments produced adventitious shoots. These regenerated shoots were initially formed as stunted rosette cluster forms and were encouraged to elongate to produce distinct shoots by transfer onto MS medium containing 0.5 mg L^?1 BA and 0.05 mg L^?1 NAA; the resulting mean number of adventitious shoots per explant was 5.81?±?0.36. The elongated shoots were readily induced to root (i.e. 89.3% of shoots) by incubation on ½-strength MS medium supplemented with 0.1 mg L^?1 IBA. This is the first report of an efficient in vitro shoot regeneration protocol for P. acerifolia through direct organogenesis using cotyledon explants. Hence, this provides a more efficient basis for the Agrobacterium-mediated genetic transformation of Platanus than previously available.     

Cryopreservation of an endangered <Emphasis Type="Italic">Hladnikia pastinacifolia</Emphasis> Rchb. by shoot tip encapsulation-dehydration and encapsulation-vitrification

The objective of the present study was the cryopreservation of monotypic endemic Hladnikia pastinacifolia Rchb. shoot tips from an in vitro culture, via encapsulation-dehydration (ED) or encapsulation-vitrification (EV). For all tested genotypes, the highest rates of shoot regrowth and multiplication were obtained after overnight preculture in 0.4 M sucrose, encapsulation in Murashige and Skoog (MS) medium with 0.4 M sucrose and 1 M glycerol, followed by polymerization in 3% (w/v) Na-alginate in MS with 0.4 M sucrose. Optimal osmoprotection was achieved for ED with 0.4 M sucrose plus 1 M glycerol and for EV with 0.4 M sucrose plus 2 M glycerol. The best dehydration time for ED was 150 min in a desiccation chamber with silica gel, and the best vitrification time for EV was 85 min in plant vitrification solution 2 (PVS2). For ED, dehydration for 150 min resulted in explant water content of 22%. When the encapsulation method was combined with ED, 53% regrowth was achieved, and when it was combined with EV, 64% regrowth was achieved. Both methods could become applicable for the long-term cryopreservation of H. pastinacifolia germplasm, although EV was faster and resulted in better final regrowth success. Genetic stability analysis of cryopreserved plant samples was carried out for two genotypes, using random amplified polymorphic DNA (RAPD) markers to compare the two different cryopreservation protocols. Significant genetic differences between the genotypes were detected and a low level of genomic variation was observed.     

Homoharringtonine production by endophytic fungus isolated from <Emphasis Type="Italic">Cephalotaxus hainanensis</Emphasis> Li

Homoharringtonine (HHT), a natural plant alkaloid derived from Cephalotaxus, has demonstrated to have a broad antitumor activity and efficacy in treating human chronic myeloid leukemia. An alternative source is required to substitute for the slow-growing and scarce Cephalotaxus to meet the increasing demand of the drug market. The objective of this study was to screen HHT-producing endophytic fungi from Cephalotaxus hainanensis Li. By screening 213 fungal isolates obtained from the bark parts of Cephalotaxus hainanensis Li, one isolate was found to be capable of biosynthesizing HHT. The fungus was identified as Alternaria tenuissima by morphological characteristics and internal transcribed spacer (ITS) sequence analysis and was named as CH1307. HHT obtained from CH1307 was analyzed through the HPLC and LC–MS/MS and NMR spectroscopy. The extract of the fermentation broth of CH1307 showed antiproliferative activities against K562 (chronic myelocytic leukemia), NB4 (acute promyelocytic leukemia), and HL-60 (promyelocytic leukemia) human cancer cell lines with IC50 values of 67.25 ± 4.26, 65.02 ± 4.75, and 99.23 ± 4.26 μg/mL, respectively. The findings suggest that HHT-producing endophytic fungus, Alternaria tenuissima CH1307 might provide a promising source for the research and application of HHT.     

Influence of Agrobacterium rhizogenes strains on hairy root induction and ‘bacoside A’ production from Bacopa monnieri (L.) Wettst.

Stable lines of hairy roots were established from leaf explants of Bacopa monnieri using different strains (A4, R1000, SA79, MTCC 532 and MTCC 2364) of Agrobacterium rhizogenes. The efficiency of hairy roots induction of these strains varied significantly and the maximum transformation frequency (75 %) was observed in case of strain SA79 using leaf explants followed by internode (55 %) in the presence of acetosyringone. Different parameters such as cell density of Agrobacterium suspension, co-cultivation period and infection time influenced the root induction frequency. Maximum frequency of root induction was obtained with bacterial density of 0.6 OD₆₀₀, 2 days of co-cultivation period and 10 min of infection time. Integration of T-DNA in the genome of hairy roots was confirmed by PCR amplification of rolB gene. Elimination of Agrobacterium from the established root cultures was ascertained by amplifying the DNA fragment specific to 16S rDNA and virD gene. All lines of hairy roots except strain A4 induced showed higher growth rate and accumulated higher levels of ‘bacoside A’ than the untransformed roots. Maximum biomass accumulation (6.8 g l^?1) and ‘bacoside A’ content (10.02 mg g^?1 DW) were recorded in case of the hairy root line induced by strain MTCC 2364.     

Assessment of genetic fidelity of <Emphasis Type="Italic">Dioscorea bulbifera</Emphasis> L. and <Emphasis Type="Italic">Dioscorea hirtiflora</Emphasis> Benth. and medicinal bioactivity produced from the induced tuberous roots

Wild tubers of Dioscorea bulbifera (Db) and Dioscorea hirtiflora (Dh) mainly used as sources of famine food and in herbal preparations are often indiscriminately collected in Africa and Asia. Therefore, there is the need to complement wild sourcing of the tubers to promote their conservation. The present study reports in vitro tuberous induction (80%) for the first time from Dh cultured on MS?+?NAA (2.5 mg/L) with IC₅₀ of 472.5?±?1.77 µg/mL using DPPH, whereas tuberous root (60%) from Db on MS?+?Kn (2.5 mg/L)?+?NAA (0.25 mg/L) had IC₅₀ of 26.97?±?1.00 µg/mL. Genetic fidelity assessment of in vitro plants compared to the wild plants revealed similar amplicon size of amplified DNA using trnH–psbA and rbcL. Similarly, micromorphological diagnostic features like oil gland, crystals (raphides), trichome and stomata type were obtained from the epidermal peels of the wild and in vitro plants. The ethyl acetate (EtOAc) extract of the flesh of Dh (wild) had the highest catechin content (108.3?±?0.69 µg/g DW). Protocatechuic acid was highest in the methanol (MeOH) extract of the flesh of Dh (0.42?±?0.02 µg/g DW), while it was detected in trace amount in the in vitro tuberous roots of MeOH extracts of Dh treated with NAA. The in vitro protocol developed in this study could be employed to multiply Dioscorea bulbifera L. and Dioscorea hirtiflora Benth. to offer genetically stable clones for the optimization of bioactive compounds and germplasms conservation.     

Agitated shoot cultures of <Emphasis Type="Italic">Aronia arbutifolia</Emphasis> and <Emphasis Type="Italic">Aronia</Emphasis> × <Emphasis Type="Italic">prunifolia</Emphasis>: biotechnological studies on the accumulation of phenolic compounds and biotransformation capability

In methanolic extracts of the biomass from agitated cultures of Aronia arbutifolia and Aronia?×?prunifolia grown on four variants of the Murashige and Skoog (MS) medium, with different concentrations of plant growth regulators (PGRs): BA and NAA (0.5–3.0 mg/l), the quantities of phenolic acids (19 compounds) and flavonoids (11 compounds) were estimated using the LC-DAD method. The amounts of individual metabolites and total contents were dependent on the concentration of PGRs in MS medium variant. The maximum total amounts of phenolic acids and flavonoids reached 360.80 and 65.26 mg/100 g DW, and 659.51 and 78.34 mg/100 g DW for A. arbutifolia and A. × prunifolia, respectively. The main metabolites in the biomass of both plants were chlorogenic acid, rosmarinic acid and quercitrin (max. 175.94, 147.98 and 41.14 mg/100 g DW, and 260.34, 225.26 and 78.34 mg/100 g DW, respectively). The cells of both plants convert the exogenously supplied hydroquinone into its β-D-glucoside—arbutin. The maximal total content of the product accumulated in the biomass and media reached 83.55 and 73.62 mg/g DW. The obtained results demonstrated for the first time a high biosynthetic potential of agitated cultures of both plants.     

Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Vigna mungo</Emphasis> using immature cotyledonary-node explants and phosphinothricin as the selection agent

Herbicide (Basta®)-tolerant Vigna mungo L. Hepper plants were produced using cotyledonary-node and shoot-tip explants from seedlings germinated in vitro from immature seeds. In vitro selection was performed with phosphinothricin as the selection agent. Explants were inoculated with Agrobacterium tumefaciens strain LBA4404 (harboring the binary vector pME 524 carrying the nptII, bar, and uidA genes) in the presence of acetosyringone. Shoot regeneration occurred for 6 wk on regeneration medium (MS medium with 4.44 μM benzyl adenine, 0.91 μM thidiazuron, and 81.43 μM adenine sulfate) with 2.4 mg/l PPT, explants being transferred to fresh medium every 14 d. After a period on elongation medium (MS medium with 2.89 μM gibberellic acid and 2.4 mg/l PPT), β-glucuronidase-expressing putative transformants were rooted in MS medium with 7.36 μM indolyl butyric acid and 2.4 mg/l PPT. β-Glucuronidase expression was observed in the primary transformants (T₀) and in the seedlings of the T₁ generation. Screening 128 GUS-expressing, cotyledonary-node-derived, acclimatized plants by spraying the herbicide Basta® at 0.1 mg/l eliminated nonherbicide-resistant plants. Southern hybridization analysis confirmed the transgenic nature of the herbicide-resistant plants. All the transformed plants were fertile, and the transgene was inherited by Mendelian genetics. Immature cotyledonary-node explants produced a higher frequency of transformed plants (7.6%) than shoot-tip explants (2.6%).     

Assessing biodegradation of furfuryl alcohol using <Emphasis Type="Italic">Pseudomonas putida</Emphasis> MTCC 1194 and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> MTCC 1034 and its kinetics

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ _max ?=?0.23 h^?1, K _s ?=?23.93 mg/l and K _i ?=?217.1 mg/l and for P. aeruginosa were determined as µ _max ?=?0.13 h^?1, K _s ?=?21.3 mg/l and K _i ?=?284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.     

Genetic fidelity assessment of <Emphasis Type="Italic">in vitro</Emphasis>-regenerated plants of <Emphasis Type="Italic">Albizia julibrissin</Emphasis> using SCoT and IRAP fingerprinting

A protocol was established for callus induction and plant regeneration of Albizia julibrissin Durazz., a multipurpose tree. Calli were induced on hypocotyl explants excised from 10- to 14-d-old in vitro seedlings cultured on Murashige and Skoog (MS) medium supplemented with α-naphthaleneacetic acid (NAA) alone or in combination with 6-benzylaminopurine (BA) or 6-furfurylaminopurine (kinetin). The highest frequency of organogenic callus (82.2?±?3.6%) was obtained on MS medium with 10.8 μM NAA and 4.4 μM BA. Calli were then cultured on MS medium with BA or zeatin, singly or in combination, for shoot regeneration. Calli cultured on MS medium with 13.2 μM BA and 4.6 μM zeatin produced the highest frequency of adventitious shoot regeneration (75.3?±?6.3%). Maximum rooting of shoots (73.3?±?5%) was achieved using half-strength MS medium with 4.9 μM indole-3-butyric acid. The genetic fidelity of 12 plants acclimatized to the greenhouse was assessed based on analyses of start codon targeted (SCoT) polymorphism and inter-retrotransposon amplified polymorphism (IRAP). The 14 SCoT and 7 IRAP adapted primers produced 71 and 34 scoreable fragments, of which 33 (46%) and 12 (35%) were polymorphic, respectively. The in vitro-raised plants exhibited 0.129–0.438 genetic distance from the mother plant and 0.000–0.788 distance from one another according to the SCoT and IRAP analyses. Although the culture method described here may not be suitable for clonal propagation of elite genotypes, it can be used for conservation of this plant.