Regular Subculturing Research Articles

Development of efficient and sustainable droplet-vitrification cryoconservation protocol for shoot tips for long-term conservation of Dahlia germplasm

Dahlias are the one amongst the tuberous herbaceous plants, valued for their attractive colorful flowers with varied size cultivated due to its ornamental value as a potted plant or cut flower in many countries. Dahlia germplasm was being conserved in the in vitro Genebank of ICAR- NBPGR, New Delhi since 2005–06 under normal growth and slow growth with a regular subculturing in every 6 months and 1 year respectively, which is laborious and time-consuming. Hence, in the current study we attempted to develop an efficient droplet-vitrification cryopreservation protocol for safe and long-term conservation of dahlia germplasm. Significant effect of type and position of explants, plant vitrification solution-2 (PVS2) dehydration duration, pregrowth period and loading solution treatment duration on post-thaw regrowth after cryoconservation was observed. Shoot tips, each being 1.0 mm in length and 0.5 mm in width positioned at the tip of in vitro shoots with a similar developmental stage (similar shoot length) were excised from in vitro cultures pregrown for 6 wks on B5 basal medium supplemented with 0.5 mg/l BAP, 30 mg/l silver nitrate and 3% sucrose. The excised shoot tips were precultured on MS + 0.3 M sucrose for overnight followed by loading solution (2.0 M glycerol and 0.4 M sucrose in liquid MS medium) treatment for 80 min, PVS2 dehydration for 30 min and then transferred onto PVS2 droplets on aluminum foil strips and cryostored in liquid nitrogen (LN). An average of 44.44% post-thaw regrowth was obtained in six accessions tested and no significant difference among the accessions was observed. There was no significant loss in regrowth of cryostored shoot tips after 1 year and 6 years of cryobanking. This justifies the genotype-wide and sustainability of the protocol for cryoconservation of Dahlia germplasm using droplet-vitrification cryopreservation procedure in the cryobanks. To the best of our knowledge, this is the first report on cryoconservation of Dahlia germplasm.

Assessment of the recovery and photosynthetic efficiency of Breviolum psygmophilum and Effrenium voratum (Symbiodiniaceae) following cryopreservation.

Many strains of Symbiodiniaceae have been isolated and their genetics, taxonomy, and metabolite production studied. Maintaining these cultures requires careful and regular sub-culturing that is costly with a high risk of species contamination or loss. Cryopreservation is a viable alternative for their long-term storage; however, there is uncertainty as to whether cryopreservation impacts the photosynthetic performance of Symbiodiniaceae. We investigated the growth rates and photosynthetic efficiency of two species, Breviolum psygmophilum and Effrenium voratum before and after cryopreservation. Rapid light curves (RLCs) produced using Pulse Amplitude Modulated (PAM) fluorometry were used to generate detailed information on the characteristics of photosystem II (PSII). The maximum electron transport rate (ETRmax) and the quantum yield (Fv/Fm) of the control (non-cryopreserved) and cryopreserved culture isolates were assessed across the growth cycle. The non-cryopreserved isolate of B. psygmophilum had a higher quantum yield than the cryopreserved isolate from day 12 to day 24, whereas there were no differences from day 28 to the late stationary phase. There were no significant differences in ETRmax. No significant differences were observed in quantum yield or ETRmax between the control and cryopreserved E. voratum isolates. The ability of cryopreserved strains to recover and regain their photosynthetic efficiency after freezing demonstrates the utility of this method for the long-term storage of these and other Symbiodiniaceae species.

Synergistic effect of copper, silver nanoparticles and hydrogen peroxide on Xanthomonas axonopodis pv. punicae

Bacterial blight disease of pomegranate caused by Xanthomonas axonopodis pv. punicae decrease crop productivity. X. axonopodis was isolated from infected fruits, stem and leaves collected from Pravara area on NAS medium. Pure culture was obtained by regular subculture and maintained on NAS medium. Trichoderma harizanium fungal biomass was generated for nanoparticle synthesis on liquid PDA medium. The aqueous cell free culture filtrate (CFCF) obtained by filtration. Nanoparticles were synthesized using 50 ml of aqueous CFCF in flasks by stirring appropriate amount of, CuSO4 (1–2 mM) solution in dark at 45 °C. Silver nanoparticles were synthesized using Trichoderma viride. SEM analysis proved silver nanoparticle size in the range 5–10 µm. Spread plate and colony count techniques proved significant antibacterial activity of silver nanoparticles against Xanthomonas axonopodis.

Somatic Embryogenesis and Plant Regeneration in Viola canescens Wall. Ex. Roxb.: An Endangered Himalayan Herb.

Viola canescens Wall. ex. Roxb. is an important but threatened medicinal herb found at 1500–2400 m above mean sea level in the Himalayas. Overexploitation and habitat preference have put the plant under serious threat. Thus, the present study was undertaken to develop an efficient protocol for in vitro propagation via somatic embryogenesis. The results revealed that plant can be regenerated successfully through somatic embryogenesis using leaf derived calli. Regular subculturing of calli on Murashige and Skoog (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D)/indole-3-butyric acid (IBA)/kinetin (Kn) and varying combinations of 2,4-D+Kn induced somatic embryogenesis. The maximum average number of somatic embryos (SE) (19.15 ± 2.66) was induced on the medium with 0.15 + 0.05 mg L−1 of 2,4-D and Kn, respectively, and this medium was used as a control. To enhance somatic embryo induction, the control MS medium was supplemented with l-glutamine (200–400 mg L−1) and casein hydrolysate (1–4%). The maximum average number of SE (27.66 ± 2.67) and average mature SE (13.16 ± 3.48) were recorded on the medium having 2 % l-glutamine and 50 mg L−1 casein hydrolysate. The induced SE were asynchronous, so, to foster their maturation, the culture medium (free from growth regulators) was supplemented with abscisic acid (ABA) and silver nitrate (AgNO3). The maximum average number (35.96 ± 3.68) of mature SE was noticed on MS medium supplemented with 1.5 mg L−1 ABA. Mature embryos had two well-developed cotyledons and an elongated hypocotyl root axis. The development of SE into plantlets was significant for embryos matured on the medium with AgNO3 and ABA, with 86.67% and 83.33% conversion on the medium with 0.20 mg L−1 6-benzylaminopurine (BAP). The plantlets thus produced acclimatized in a growth chamber before being transferred to the field, which showed 89.89% survival. The plants were morphologically similar to the mother plant with successful flowering.

Rapid Lentiviral Vector Producer Cell Line Generation Using a Single DNA Construct

Stable suspension producer cell lines for the production of vesicular stomatitis virus envelope glycoprotein (VSVg)-pseudotyped lentiviral vectors represent an attractive alternative to current widely used production methods based on transient transfection of adherent 293T cells with multiple plasmids. We report here a method to rapidly generate such producer cell lines from 293T cells by stable transfection of a single DNA construct encoding all lentiviral vector components. The resulting suspension cell lines yield titers as high as can be achieved with transient transfection, can be readily scaled up in single-use stirred-tank bioreactors, and are genetically and functionally stable in extended cell culture. By removing the requirement for efficient transient transfection during upstream processing of lentiviral vectors and switching to an inherently scalable suspension cell culture format, we believe that this approach will result in significantly higher batch yields than are possible with current manufacturing processes and enable better patient access to medicines based on lentiviral vectors.

A cell suspension based uptake method to study high affinity glucosinolate transporters

BackgroundGlucosinolates are an important class of secondary metabolites characteristic to the order Brassicales. They are known to play a major role in plant defense and from the human perspective, can be anticarcinogenic or antinutritive. GTRs are plasma-membrane localized high affinity glucosinolate transporters, which are important components of the source (leaf) to sink (seed) translocation of intact glucosinolates in members of Brassicaceae family. GTRs are identified as major candidates for Brassica crop improvement, thus dictating a need for their functional characterization. However, currently there are limitations in availability of heterologous assay systems for functional characterization of plant secondary metabolite transporters. To date, the animal-based Xenopus oocyte system is the best established heterologous system for functional characterization of these transporters. Inherent biochemical and physiological attributes unique to the plant membranes necessitate the need for developing plant-based transporters assay systems as well.MethodsIn this study, Agrobacterium mediated transformation was used to develop GTR expressing cotton cell lines (CCL-1) for functional characterization of the Arabidopsis high affinity glucosinolate transporters, AtGTR1 and AtGTR2. Following sub-cellular localization of AtGTRs, we standardized the glucosinolate uptake assays using cell suspension cultures of AtGTR expressing CCL-1 its requirement of pH, salt, and time based glucosinolate uptake. Using the GTR expressing CCL-1, we subsequently performed kinetic analysis of AtGTR1 and AtGTR2 for different glucosinolate substrates, sinigrin, gluconapin and sinalbin.ResultsSeveral clones expressing each of AtGTR1 and AtGTR2 were obtained showing high level of GTR expression and were maintained through regular sub-culturing. Both AtGTR1 and AtGTR2 are predominantly plasma-localized proteins when overexpressed in CCL-1 cells. Uptake assays were standardized, suggesting that glucosinolate uptake of GTR expressing CCL-1 is robust within the physiological pH range 5–6, and at lower concentration of nitrate salts. GTR expressing CCL-1 cells show increasing glucosinolate accumulation in time course experiment. Kinetic studies over a wide glucosinolate concentrations (10–800 µM) revealed that our novel assay system displayed robust GTR-mediated uptake of different glucosinolates and unambiguously helps elucidate the saturable kinetics of GTRs. Our system confirms the high affinity of AtGTRs for both aliphatic and aromatic glucosinolates.ConclusionThe transporter assay system described in this study holds potential for studying sub-functionalization amongst GTR homologs present across Brassicaceae family. The fast growing CCL-1 cells, confer the benefits of an in vitro system for quick assays and is plant based thus enabling optimal expression without sequence modifications. The efficient functioning of the GTR transporters in the heterologous CCL-1 opens the possibility of using this plant cell suspension system for functional characterization of other metabolite transporters.

Disappearance of Quorum Sensing in Burkholderia glumae During Experimental Evolution.

The plant pathogen Burkholderia glumae uses quorum sensing (QS) that allows bacteria to share information and alter gene expression on the basis of cell density. The wild-type strain of B. glumae produces quorum-sensing signals (autoinducers) to detect their community and upregulate QS-dependent genes across the population for performing social and group behaviors. The model organism B. glumae was selected to investigate adaptation, estimate evolutionary parameters, and test diverse evolutionary hypotheses by using experimental evolution. The wild-type B. glumae virulent strain showed genotypic changes during regular subculture due to oxygen limitation. The laboratory-evolved clones failed to produce the signaling molecule of C8-HSL/C6-HSL for activation of the quorum-sensing system. Further, the laboratory-evolved clones failed to produce catalase and oxalate for protecting themselves from the toxic environment at stationary phase and phytotoxins (toxoflavin) for infecting rice grain, respectively. The laboratory-evolved clones were completely sequenced and compared with the wild-type. Sequencing analysis of the evolved clones revealed that mutations in QS-responsible genes (iclR), sensor genes (shk, mcp), and signaling genes (luxR) were responsible for quorum-sensing activity failure. The experimental results and sequencing analysis revealed quorum-sensing process failure in the laboratory-evolved clones. In conclusion, the wild-type B. glumae strain was often exposed to oxidative stress during regular subculture and evolved as an avirulent strain (quorum-sensing mutant) by losing the phenotypic and genotypic characteristics.

A robust micropropagation protocol for genetically true to type plants of Phule Arakta pomegranate

A rapid and reproducible micropropagation protocol for pomegranate cv. Phule Arakta has been developed using nodal segments of field grown plant. Bud break was induced in basal Woody Plant Medium (WPM) as well as compared when WPM was supplemented with plant growth regulators. Multiple shoot proliferation was induced in the nodal segments on WPM fortified with different concentration of benzyladenine (BAP) where 2mg/l BAP developed maximum number of shoots. Elongation of shoots was further amplified with the addition of adjuvant silver nitrate. Browning of culture medium was controlled by the addition of polyvinylpyrrolidone (PVP) and regular sub-culturing enhanced shoot multiplication as well as elongation. Rooting was induced in the regenerated shoots using Indole-3-butyric acid (IBA) and 3-Indoleacetic acid where best result was obtained using shock treatment with NAA. Sub-culturing resulted in denser and better rooting. The rooted plantlets were further acclimatized and then established in soil. The clonal fidelity of the in vitro grown cultures was assessed using Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeats (ISSR) markers. The 10 RAPD decamers produced 55 bands and 4 ISSR produced 19 bands in total. RAPD primers OPC-08, OPC-13 and OPD-07 produced the highest number of distinct bands and ISSR primer UBC-834 produced maximum distinct bands. All the bands were monomorphic which confirms the genetic fidelity of the in-vitro raised P. granatum cv. Phule Arakta and supported the method of mass production of true to type progenies using tissue culture.

Diverse cellular colonizing endophytic bacteria in field shoots and in vitro cultured papaya with physiological and functional implications.

The study was envisaged to assess the extent of normally uncultivable endophytic bacteria in field papaya plants and in vitro established cultures adopting cultivation vs molecular analysis and microscopy. Surface-sterilized axillary shoot-buds of papaya 'Arka Surya' revealed high bacterial diversity as per 16S rRNA metagene amplicon sequencing (6 phyla, 10 classes, 21 families) with an abundance of Pseudomonas (Gammaproteobacteria), which also formed a common contaminant for in vitro cultured field explants. Molecular analysis of seedling shoot-tip-derived healthy proliferating cultures of three genotypes ('Arka Surya', 'Arka Prabhath', 'Red Lady') with regular monthly subculturing also displayed high bacterial diversity (11-16 phyla, >25 classes, >50 families, >200 genera) about 12-18 months after initial establishment. 'Arka Surya' and 'Red Lady' cultures bore predominantly Actinobacteria (75-78%) while 'Arka Prabhath' showed largely Alphaproteobacteria corroborating the slowly activated Methylobacterium sp. Bright-field direct microscopy on tissue sections and tissue homogenate and epi-fluorescence microscopy employing bacterial DNA probe SYTO-9 revealed abundant intracellular bacteria embracing the next-generation sequencing elucidated high taxonomic diversity. Phylogenetic investigation of communities by reconstruction of unobserved states- PICRUSt- functional annotation suggested significant operational roles for the bacterial-biome. Metabolism, environmental information processing, and genetic information processing constituted major Kyoto Encyclopedia of Genes and Genomes KEGG attributes. Papaya stocks occasionally displayed bacterial growth on culture medium arising from the activation of originally uncultivable organisms to cultivation. The organisms included Bacillus (35%), Methylobacterium (15%), Pseudomonas (10%) and seven other genera (40%). This study reveals a hidden world of diverse and abundant conventionally uncultivable cellular-colonizing endophytic bacteria in field shoots and micropropagating papaya stocks with high genotypic similarity and silent participation in various plant processes/pathways.

Evaluation of Probiotic Characteristics of Lactic Acid Bacteria Isolated from Two Commercial Preparations Available in Indian Market.

The enormous health benefits associated with probiotics has resulted in an increased consumption of probiotic supplements. Several factors like regular sub-culturing, storage, unfavourable conditions etc. might compromise the efficacy and/or safety of lactic acid bacteria which are the major components of many probiotic preparations available in the market. The present study evaluated the probiotic characteristics and safety of probiotic bacteria isolated from two preparations available commercially in India. The products did not specify the genera, species or strains of the bacteria used. These were cultured using standard microbiological methods for cultivation of lactic acid bacteria. Bacteria were identified by PCR amplification and sequence analysis of 16S rRNA gene. Microbiological and molecular analyses revealed that both preparations contained homogenous population of Enterococcus faecium and Pediococcus acidilactici respectively. Assessment for several essential and desirable probiotic properties revealed that both the probiotic strains were safe and resistant to salt, lysozyme, bile salt and common antibiotics. The probiotic preparation containing P. acidilactici was better than that containing E. faecium as it survived in low pH and showed bile salt hydrolase activity. The probiotic preparation containing P. acidilactici also exhibited cholesterol-lowering activity.

Efficient Technique for Long-Term <i>in Vitro</i> Storage of Transgenic Aspen Genotypes

In vitro culture of isolated cells from tissues and organs is sometimes used to preserve and reproduce unique genotypes of woody plants. The technique, however, requires regular subculturing which raises storage costs and creates risks for contamination and accumulation of somaclonal variations. We examined the effects of sugar composition of culture medium, the length of photoperiod, light intensity, and ambient temperature on the survival of plant material in vitro. The study was performed on 49 genotypes of Populus tremula (46 transgenic genotypes carrying GFP-, Xeg- and Gus-genes, and 3 control (wild-type) genotypes). It was shown that effective storage of plants was achieved through optimization of the combined effects of all storage parameters under study. Based on the experimental data, we developed a protocol for long-term in vitro storage of desirable genotypes without subculture and with a survival rate of up to 98%. The best results were obtained when the plant material was pre-cultured on a WPM medium containing 15 g/L sucrose, 7.5 g/L sorbitol and 7.5 g/L mannitol, and then stored at +4°C under a 24-hour light day cycle with only 8 hours of light per day and maximum light intensity of 2000 lux. Post-storage recovery was done by culturing on a medium containing 1 mg/L gibberellic acid. The developed method can be used for effective in vitro storage of the studied genotypes for up to 24 months without subculture.

Effect of l-glutamine and casein hydrolysate in the development of somatic embryos from cotyledonary leaf explants in okra (Abelmoschus esculentus L. monech)

Okra is an important vegetable crop and very few studies have been carried out for the development and in vitro regeneration of somatic embryos. In this study, we have developed an efficient protocol for somatic embryogenesis from cotyledonary leaf explants of Okra (Abelmoschus esculentus L. Monech). In this investigation, the genotype CoBhH1 of A. esculentus was tested with different amino acids and casein hydrolysate for the optimization culture conditions and development of somatic embryos. The explants were cultured on Murashige and Skoog (MS) medium containing Gamborgs (B5) vitamins, 400mg/l l-glutamine, 300mg/l casein hydrolysate, 8g agar, 1.5mg/l 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 1mg/l naphthaleneacetic acid (NAA). Proembryoids were observed after 4weeks of regular subculturing in the same media. Globular to torpedo shaped somatic embryo development was observed after 8weeks of culture. Mature embryos were selected by their cotyledonary stage and subcultured on the regeneration medium. Half strength MS medium containing B5 vitamin (half strength), 30g sucrose, 8g agar, 1mg/l BAP and 0.5mg/l GA3 were used for regeneration and shoot elongation. A total of 17.43 plantlets were obtained from single explant through this protocol. The plantlets were transferred to plastic cups in laboratory condition and finally transferred to the greenhouse with a 100% survival rate. Inter simple sequence repeats (ISSR) analysis was used to check the genetic fidelity of regenerated plants. No polymorphism was detected revealing the genetic integrity of regenerated plantlets. The regenerated plants were morphologically similar to those of the mother plant.

Seasonal and micro-environmental factors controlling clonal propagation of mature trees of marwar teak [Tecomella undulata (Sm.) Seem

A simple method has been developed for clonal propagation of mature trees of Tecomella undulata (Sm.) Seem, a medicinally important deciduous timber tree of hot arid regions, via multiple shoot proliferation from axillary buds after examining the role of season influences and physico–chemical conditions on micropropagation. Spring season (March–April) was the best period for contamination free establishment of explants and maximum sprouting of healthy axillary buds. Shoots proliferated directly from the explant nodes cultured on Murashige and Skoog’s medium containing cytokinins, BAP supporting better growth compared to kinetin during shoot induction as well as multiplication phase. Cytokinin concentration influenced the bud induction frequency and optimal response of 2.6 buds per explant was achieved in 86.66% explants on media supplemented with 10 µM BAP. Stunted shoot buds with excessive callus were observed when cytokinin concentration was increased beyond optimal levels. Ascorbic acid (50 mg/l), arginine and citric acid (25 mg/l each) were added to proliferation and multiplication media for reducing callus proliferation and better shoot growth. Among the media (B5, MS, NN, WPM and SH) tested, SH was best for shoot multiplication. Shoot cultures were multiplied by regular subculture of axillary shoots on SH medium containing 5.0 µM each of BAP and kinetin. Shoots produced roots when cultured on ½× SH medium + 10 μM IBA. Regenerated plantlets were successfully transferred to field after hardening and acclimatization. Genetic homogeneity of tissue culture raised plants was confirmed by generation of monomorphic DNA fragments with Start codon targeted and intersimple sequence repeat (ISSR) markers.

An efficient and economic method for in vitro propagation of Arnebia euchroma using liquid culture system

Arnebia euchroma Royle (Johnston) (Boraginaceae), a rich source of shikonin derivatives, is valued for its medicinal as well as coloring properties. In vitro multiplication of this plant species is important for continuous supply of raw material for pharmaceutical industries as well as to conserve the germplasm of this rare and endangered species. The present work assessed the use of liquid (shake flasks and static) medium for its potential to induce shoot growth and multiplication using agar-gelled medium as a control. Shoot cultures were initiated from rhizome buds on Murashige and Skoog (MS) agar-gelled medium with 6-benzylaminopurine (BAP; 10.0 µM) and α-naphthalene acetic acid (NAA; 5.0 µM) and maintained by regular sub-culturing on Kinetin (Kn; 5.0 µM) containing medium. Best response in terms of shoot proliferation was observed in liquid shake flask cultures. MS medium supplemented with Thidiazuron (TDZ; 1.0 µM) favored 14-fold increase in shoot number followed by BAP (10.0 µM; 11 fold) and Kn (2.5 µM; 9 fold). Initial culturing in liquid shake flask with TDZ and a periodic transfer to agar-gelled medium having Kn was beneficial for sustained production of normal multiple shoots. An efficient and economic protocol was developed using liquid culture system for in vitro propagation of A. euchroma. The present protocol of in vitro shoot multiplication could be utilized for further studies related to large scale propagation as well as conservation of this plant species.

Tanshinones in Culture of Salvia Przewalskii Maxim in Vitro

Abstract The content of four tanshinones was determined in different in vitro cultures of Salvia przewalskii. Accumulation of tanshinones depended on the type and age of tissue and could be altered by growth conditions. Differentiated tissues (in vitro cultured shoots, shoots and roots of plantlets regenerated in vitro) contained more diterpenoids than undifferentiated tissues (i.e., the four callus lines). Root was the most important organ for tan-shinone accumulation. The highest levels were achieved in roots of 4-week-old plantlets (5.1-5.6 mg g−1 DW); the shoots used for root induction were maintained on multiplication medium for 2.5-4 years with regular subcultures every 4 weeks.

Cloudberry (Rubus chamaemorus) cell culture with bioactive substances: Establishment and mass propagation for industrial use

The availability and quality of wild berry material is strongly affected by seasonal variations, human resources, and both chemical and biological pollutants. Here we describe for the first time the industrial scale biotechnological production of cloudberry (Rubus chamaemorus) cells of consistent quality and defined chemical composition. Callus was initiated from sterile cuts of the wild cloudberry leaves. Bright yellow, soft callus of uniform quality was selected by regular subculturing, and a stable line successfully used for suspension culture was obtained within 18 months. The cultivation process of the cell culture was initiated in 250 mL shake flasks and stepwise expanded to a stirred tank bioreactor of 300 L working volume. Each growth period in flasks and fed‐batch cultures in bioreactors was 10 ± 3 days. Viability of the cultures was approximately 90% throughout the cultivation processes. The 300 L culture was harvested by pressure filtration and the biomass was freeze‐dried. The 300 L yields of fresh cells and freeze‐dried material were 19 and 2 kg, respectively. The unusual phenolic profile of cloudberry cells including flavanols, as well as their fatty acid composition with a high proportion of α‐linolenic acid and high protein content, make them a unique and interesting alternative for industrial applications for various industrial fields.

Production and evaluation of transgenic sorghum for resistance to stem borer

Transgenic sorghum plants were produced through particle bombardment and Agrobacterium methods in two elite, but recalcitrant genotypes of Sorghum bicolor L. Moench. Use of target cells from developing tissues (immature embryos and multiple shoot buds), pre-culture of target tissue, small size of target tissue (2–3 mm), and regular subculture improved the selection and regeneration efficiencies. Addition of amino acid l-cysteine during co-cultivation and blotting sheet interface was helpful for complete decontamination of Agrobacterium tumefaciens and regeneration of transgenic plants. We demonstrated production of transgenic sorghum plants expressing a Bacillus thuringiensis lepidopteran toxin, through tailored in vitro protocols. Our results showed that decontamination of agrobacteria employing subtle treatments aided recovery of transgenic plants in recalcitrant genotypes. We generated 14 independent transgenic lines carrying different classes of B. thuringiensis toxin genes, cry1Aa and cry1B. Many single copy events were generated in two elite parental lines, CS3541 and 296B. Accumulation of the B. thuringiensis protein in leaves during the susceptible period of plant growth ranged from 35 to 500 ng/g fresh leaf tissue. Comprehensive insect bioassays for tolerance to spotted stem borer (Chilo partellus) were conducted through leaf disk and whole plant assays. Transgenic progeny plants showed 20–30% of damage as compared to 70–80% in non-transformed controls.

Studying the Possible Biotransformation of the Cytotoxic Diterpenoid Paclitaxel Using Jatropha Curcas Cell Suspension Culture

Aims: To establish a cell suspension culture of Jatropha curcas, family Euphorbiaceae for the biotransformation of the diterpenoid anti-cancer compound paclitaxel. Study Design: The development and maintenance of callus lines from the leaves and petioles of J. curcas and the study of the culture growth curve followed by the inoculation of the callus with paclitaxel as the substrate and monitoring of the culture viability as well as substrate biotransformation. Methodology: Explants from the leaves and petioles of J. curcas were prepared and placed on MS supplemented media for callus induction and maintained by regular subculturing until a stable callus line was achieved. Some of the callus tissue was transferred to MS liquid medium to produce cell suspension cultures. Three sets of cell suspension cultures were established for 5, 10 and 14 days respectively under the same conditions. Paclitaxel (5 mg) was administrated to each flask and incubated for 3, 6 and 10 days. Results: A callus line of J. curcas leaves and petioles was maintained successfully over a Research Article European Journal of Medicinal Plants, 3(2): 241-253, 2013 242 period of two consecutive years without any change in growth rate. A stable cell suspension culture was developed using the obtained callus and maximum increase in fresh weight was reached on day 21 which was 5-6 fold over initial fresh weight. The cell suspension cultures were inoculated with the diterpenoid paclitaxel (5 mg) at different time intervals through the growth cycle phases. The incubation of paclitaxel proved that the cell culture biotransformation capability could not affect the paclitaxel molecular structure and that the applied dose of paclitaxel was not cytotoxic to the cell cultures up to 18 days of incubation. Conclusion: This is the first report of a biotransformation trial using J. curcas cell culture and results obtained should be considered when using J. curcas cell line for terpenoid biotransformation.

Effect of auxins on axillary and de novo shoot regeneration from in vitro shoot cultures derived from forced epicormic buds of teak (Tectona grandis L.)

In this presentation, we report on de novo and axillary shoot regeneration and rooting of shoots maintained over a long term, from cultures of Tectona grandis L. Shoot-tips of teak shoots forced from epicormic buds were used as the starting material for axenic shoot-culture establishment. Long term maintenance of such axenic shoot cultures was carried out by regular sub-culturing on MS media supplemented with N6-benzyleadenine (BA, 8.8 μmol·L−1) and indole-3-butyric acid (IBA, 2 μmol·L−1) for 24 months. Vigorously growing shoot tips (2–3 cm long) were inoculated on the MS basal medium supplemented with different concentrations (0, 1, 2, 4, 6, 8 or 10 μmol·L−1) of either IBA or α-naphthaleneacetic acid (NAA) for rooting. Axillary and de novo shoots were developed from axillary and cut basal ends of shoots, respectively. Shoots growing on auxins were further sub-cultured (every 15 days) and maintained for 45 days. The greatest number of de novo (5.06) as well as axillary shoots (2.85) was observed on the MS medium supplemented with 10 μmol·L−1 NAA or 8 μmol·L−1 IBA, respectively, after 45 days. The combinations of both IBA (μmol·L−1) + NAA (μmol·L−1) were tested at different concentrations (4 + 4, 6 + 6, 8 + 8) supplemented to a half strength MS basal medium with 0.1% activated charcoal for rooting of decapitated and non-decapitated de novo and axillary shoots. Rooting from non-decapitated de novo shoots was highest (93.33%) with a mean number of roots of 4.61 on this medium, supplemented with 6 μmol·L−1 IBA + 6 μmol·L−1 NAA, after 36 days of initial culture. Individual auxin, however, was not effective for root induction. Rooted shoots were acclimatized in a green house and after four weeks plantlets were transferred to the field.

Salinity-induced modulation of growth and antioxidant activity in the callus cultures of miswak (Salvadora persica).

Miswak (Salvadora persica Linn.) is a medium-sized tree, desert facultative halophytic plant. Besides edible fruits and non-edible seed oil, the plant contains several bioactive compounds like alkaloids, tannins, saponins and sterols related to food and cosmetic industries. In the present study, physiological responses and antioxidant potential under salinity stress were investigated in callus cultures of S. persica to evaluate its use as a source of antioxidant. The callus cultures were grown on MS medium supplemented with 0.5 mg/l each of 2,4,5-T and BAP, which could be established successfully by regular subcultures of slow growing callus on this medium for several months. Increased dry weight, soluble proteins, proline, soluble carbohydrates and CAT activity were recorded under NaCl stress in comparison to control cultures. The DPPH and FRAP antioxidant activities were gradually elevated in NaCl-treated callus, whereas SOD quenching was recorded maximum at 200 mM. A significant correlation between antioxidant capacity and phenol content was observed, indicating that phenolic compounds are the major contributors to the antioxidant potential in S. persica. These findings suggest that increased salinity stress caused elevated antioxidant potentials and the plants grown in such conditions may serve as potential source of antioxidant.