The effect of chitosan and ß-cyclodextrin on glucosinolate biosynthesis in Brassica rapa ssp. pekinensis (Chinese cabbage) shoot culture.

Chitosan is a polycationic polysaccharide derived from chitin, and ß-cyclodextrin is a type of macrocyclic oligosaccharide linked by α-1,4 glycosidic bonds. These compounds are recognized as effective elicitors in the biosynthesis of secondary metabolites in plants. These elicitors were studied to assess the growth of shoots and the synthesis of glucosinolates (GSLs) from elicited shoots in Chinese cabbage under controlled in vitro conditions for the first time. Chitosan at 150 mg L-1 supplemented in the optimized shoot induction recovered maximum quantities of total GSLs (7.344 µmol g-1 DW) at the end of 5th week of culture duration, followed by ß-cyclodextrin (15 mg L-1) with the synthesis of GSLs (6.379 µmol g-1 DW) at the end of 4th week of culture. The application of chitosan completely deteriorated the growth of shoots, whereas ß-cyclodextrin did not affect and even increased the growth of shoots (4.66 g DW). Upon elicitation, the individual got GSLs contents exhibited various fold changes (1.78-to-23.5-fold). Real-time PCR analysis of essential GSLs biosynthesis genes like MAM1, ST5b, AOP2, FMOGS-OX1, CYP83B1, CYP81F2, ST5a, and CYP81F4 revealed substantial higher expression upon elicitation. This present study would provide a steady production of GSLs in Chinese cabbage shoots with the influence of carbohydrate-based elicitors for pharmaceutical or food companies in the future.

Optimization of Protoplast Isolation from Leaf Mesophylls of Chinese Cabbage (Brassica rapa ssp. pekinensis) and Subsequent Transfection with a Binary Vector.

Chinese cabbage is an important dietary source of numerous phytochemicals, including glucosinolates and anthocyanins. The selection and development of elite Chinese cabbage cultivars with favorable traits is hindered by a long breeding cycle, a complex genome structure, and the lack of an efficient plant transformation protocol. Thus, a protoplast transfection-based transformation method may be useful for cell-based breeding and functional studies involving Chinese cabbage plants. In this study, we established an effective method for isolating Chinese cabbage protoplasts, which were then transfected with the pCAMBIA1303 binary vector according to an optimized PEG-based method. More specifically, protoplasts were isolated following a 4 h incubation in a solution comprising 1.5% (v/v) cellulase, 0.25% (v/v) macerozyme, 0.25% (v/v) pectinase, 0.5 M mannitol, 15 mM CaCl2, 25 mM KCl, 0.1% BSA, and 20 mM MES buffer, pH 5.7. This method generated 7.1 × 106 protoplasts, 78% of which were viable. The gfp reporter gene in pCAMBIA1303 was used to determine the transfection efficiency. The Chinese cabbage protoplast transfection rate was highest (68%) when protoplasts were transfected with the 40 µg binary vector for 30 min in a solution containing 40% PEG. The presence of gusA and hptII in the protoplasts was confirmed by PCR. The methods developed in this study would be useful for DNA-free genome editing as well as functional and molecular investigations of Chinese cabbage.

L-Cysteine Increases the Transformation Efficiency of Chinese Cabbage (Brassica rapa ssp. pekinensis).

Successful Agrobacterium-mediated transformations of Chinese cabbage have been limited owing to the plant's recalcitrant nature, genomic background and explant necrosis upon infection, which hinders the transfer of T-DNA region into the Chinese cabbage. Consequently, in the current experiment, a stable Agrobacterium tumefaciens-mediated transformation method for Chinese cabbage cv. Kenshin established by employing important anti-oxidants in the co-cultivation and subsequent regeneration media. Four-day-old in vitro derived cotyledon explants were infected with A. tumefaciens strain GV3101 harboring the vector pCAMIBA1303. Cotyledon explants exposed to an Agrobacterium suspension (OD600 of approximately 0.6) for 10 min and then incubated for 3 days co-cultivation in Murashige and Skoog medium containing an L-cysteine + AgNO3 combination exhibited the highest ß-glucuronidase (GUS) expression (94%) and explant regeneration efficiency (76%). After 3 days, the cotyledon explants were subjected to three selection cycles with gradually increasing hygromycin B concentrations (10 to 12 mg/L). The incorporation and expression of hptII in T0 transformed plants were verified by polymerase chain reaction and Southern blot analyses. These transgenic plants (T0) were fertile and morphologically normal. Using the present protocol, a successful transformation efficiency of 14% was achieved, and this protocol can be applied for genome editing and functional studies to improve Chinese cabbage traits.

Elicitation Approaches for Withanolide Production in Hairy Root Culture of Withania somnifera (L.) Dunal.

Withania somnifera (L.) Dunal is a versatile medicinal plant extensively utilized for production of phytochemical drug preparations. The roots and whole plants are traditionally used in Ayurveda, Unani, and Siddha medicines, as well as in homeopathy. Several studies provide evidence for an array of pharmaceutical properties due to the presence of steroidal lactones named "withanolides." A number of research groups have focused their attention on the effects of biotic and abiotic elicitors on withanolide production using cultures of adventitious roots, cell suspensions, shoot suspensions, and hairy roots in large-scale bioreactor for producing withanolides. This chapter explains the detailed procedures for induction and establishment of hairy roots from leaf explants of W. somnifera, proliferation and multiplication of hairy root cultures, estimation of withanolide productivity upon elicitation with salicylic acid and methyl jasmonate, and quantification of major withanolides by HPLC. The protocol herein described could be implemented for large-scale cultivation of hairy root biomass to improve withanolide production.

Involvement of exogenous polyamines enhances regeneration and Agrobacterium-mediated genetic transformation in half-seeds of soybean.

The present work demonstrates the participation of polyamines (PAs) to improve direct regeneration and Agrobacterium-mediated transformation in soybean half-seeds. The inclusion of PAs to culture medium along with optimal plant growth regulators (PGRs) enhanced shoot induction [98.3 %; 4.44 µM N6-benzyladenine (BA) and 103.27 µM spermidine] and elongation [90.0 %; 1.45 µM gibberellic acid (GA3) and 49.42 µM spermine]. The polyamine putrescine (62.08 µM) alone greatly enriched root induction (96.3 %). The influence of PAs on transformed plant production was assessed by comparing optimized protocol (comprising PAs and PGRs) with a regeneration system involving only PGRs. Plant transformation was performed in half-seeds of cultivar DS 97-12 using strain EHA105 harboring pCAMBIA1301. Transgene expression and integration was confirmed by GUS staining, PCR, and Southern hybridization. The transformed explants/materials successively cultured on co-cultivation (BA and spermidine), shoot induction (BA and spermidine), shoot elongation (GA3 and spermine), and rooting medium (putrescine) showed enhanced transformation efficiency (29.3 %) compared with its counterparts (14.6 %) with respective PGR alone [BA, GA3, or indole-3-butyric acid (IBA)]. Overall findings of the study suggest that involvement of PAs improved T-DNA transfer during co-cultivation, and delivered most suitable condition for efficient regeneration/survival, which led to enhanced transformation efficiency in soybean.

L-Dopa production and antioxidant activity in Hybanthus enneaspermus (L.) F. Muell regeneration.

Hybanthus enneaspermus is an ethanobotanical plant extensively used in Indian traditional medicine. Quick and efficient in vitro mass propagation of this plant species was established for commercial utilization from leaf and node explants using various concentrations and combinations of plant growth regulators and polyamines. The maximum number of multiple shoots per leaf explant (40 shoots) was achieved on MS medium supplemented with 20 mg/l spermidine in combination with 4 mg/l BA+1.5 mg/l IAA after 8 weeks of culture. The elongated shoots were rooted (16 roots/shoot) on MS medium with the best concentration of IBA (1.5 mg/l) and in combination with 20 mg/l putrescine after 5 weeks of culture. The plants were successfully acclimatized (98 %) in the sand: soil: vermiculite mixture (1:1:1 v/v/v) in the greenhouse. An increased antioxidant activity was recorded in vitro regenerated shoots when compared to in vitro-induced roots. L-Dopa content was recorded higher in leaves (8.31 mg/g DW) followed by stem (6.22 mg/g DW) and root (3.22 mg/g DW) of leaf-derived plants than the field-grown parent plant after 5 weeks. By adopting this protocol, the regenerated-plants could be used for drug production and pharmacology work with as an alternative to field-grown plants.

Sonication, Vacuum Infiltration and Thiol Compounds Enhance the Agrobacterium-Mediated Transformation Frequency of Withania somnifera (L.) Dunal.

In the present study, we have established a stable transformation protocol via Agrobacterium tumafacines for the pharmaceutically important Withania somnifera. Six day-old nodal explants were used for 3 day co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring the vector pCAMIBA2301. Among the different injury treatments, sonication, vacuum infiltration and their combination treatments tested, a vacuum infiltration for 10 min followed by sonication for 10 sec with A. tumefaciens led to a higher transient GUS expression (84% explants expressing GUS at regenerating sites). In order to improve gene integration, thiol compounds were added to co-cultivation medium. A combined treatment of L-Cys at 100 mg/l, STS at 125 mg/l, DTT at 75 mg/l resulted in a higher GUS expression (90%) in the nodal explants. After 3 days of co-cultivation, the explants were subjected to three selection cycles with increasing concentrations of kanamycin [100 to 115 mg/l]. The integration and expression of gusA gene in T0 and T1 transgenic plants were confirmed by polymerase chain reaction (PCR), and Southern blott analysis. These transformed plants (T0 and T1) were fertile and morphologically normal. From the present investigation, we have achieved a higher transformation efficiency of (10%). Withanolides (withanolide A, withanolide B, withanone and withaferin A) contents of transformed plants (T0 and T1) were marginally higher than control plants.

Expression of important pathway genes involved in withanolides biosynthesis in hairy root culture of Withania somnifera upon treatment with Gracilaria edulis and Sargassum wightii.

The investigation of seaweeds, Gracilaria edulis and Sargassum wightii extracts was carried out for the estimation of growth characteristics and major withanolides production in hairy root culture of Withania somnifera. The extract of G. edulis (50%) in MS liquid basal medium enabled maximum production of dry biomass (5.46 g DW) and withanolides contents (withanolide A 5.23 mg/g DW; withaferin A 2.24 mg/g DW and withanone 4.83 mg/g DW) in hairy roots after 40 days of culture with 48 h contact time. The obtained withanolides contents were significantly higher (2.32-fold-2.66-fold) in hairy root culture when compared to the control. RT PCR analysis of important pathway genes such as SE, SS, HMGR and FPPS exhibited substantial higher expression upon the seaweed extracts treatment in hairy root culture. This experiment would paw a platform for withanolides production in hairy root culture with the influence of sea weed extracts for pharmaceutical companies in the future.

Effect of carbon and nitrogen sources on in vitro flower and fruit formation and withanolides production in Withania somnifera (L.) Dunal.

We studied the influence of sucrose and nitrogen concentration on in vitro flowering and fruit setting in elongated shoots of Withania somnifera. BA (1.5 mg/l) and IAA (0.3 mg/l) on MS medium supplemented with 4% sucrose showed 67% of in vitro flower induction frequency, 9 flowers/shoot, 4 fruits/shoot and 11 seeds/fruit in elongated-shoots. Different concentrations of nitrogen sources (L-glutamine, adenine sulphate, ammonium nitrate, potassium nitrate and sodium nitrate 5-25 mg/l) were tested in combination with 4% sucrose and BA at 1.5 mg/l and IAA at 0.3 mg/l. Highest number of flowers (20 flowers/shoot; 2.2-fold) and fruits (16 fruits/shoot; 3.39-fold), fruit setting (12 seeds/fruit; 1.08-fold) at a higher frequency (88%) were achieved on MS medium augmented with 15 mg/l adenine sulphate with same PGRs and sucrose concentration. The maximum production of withanolide A (0.68 mg/g DW) and withanolide B (0.77 mg/g DW) was recorded in in vitro fruits. Highest accumulation of withaferin A (2 mg/g DW) was quantified from in vitro flowers, whereas, it was low in in vitro fruits (0.49 mg/g DW withaferin A). However, withanone (0.23 mg/g DW) was found accumulated uniformly in both in vitro flowers and fruits compared to control.

Enhanced biosynthesis of withanolides by elicitation and precursor feeding in cell suspension culture of Withania somnifera (L.) Dunal in shake-flask culture and bioreactor.

The present study investigated the biosynthesis of major and minor withanolides of Withania somnifera in cell suspension culture using shake-flask culture and bioreactor by exploiting elicitation and precursor feeding strategies. Elicitors like cadmium chloride, aluminium chloride and chitosan, precursors such as cholesterol, mevalonic acid and squalene were examined. Maximum total withanolides detected [withanolide A (7606.75 mg), withanolide B (4826.05 mg), withaferin A (3732.81 mg), withanone (6538.65 mg), 12 deoxy withanstramonolide (3176.63 mg), withanoside IV (2623.21 mg) and withanoside V (2861.18 mg)] were achieved in the combined treatment of chitosan (100 mg/l) and squalene (6 mM) along with 1 mg/l picloram, 0.5 mg/l KN, 200 mg/l L-glutamine and 5% sucrose in culture at 4 h and 48 h exposure times respectively on 28th day of culture in bioreactor. We obtained higher concentrations of total withanolides in shake-flask culture (2.13-fold) as well as bioreactor (1.66-fold) when compared to control treatments. This optimized protocol can be utilized for commercial level production of withanolides from suspension culture using industrial bioreactors in a short culture period.

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle.

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3%) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA3 [5 mg/l]) and higher direct somatic embryogenic frequency (89.6%). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA3 (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.

Transfer and targeted overexpression of γ-tocopherol methyltransferase (γ-TMT) gene using seed-specific promoter improves tocopherol composition in Indian soybean cultivars.

Soybean oil contains high levels of tocopherols which are an important source of vitamin E in human diet. The conversion of γ- to α-tocopherol catalyzed by γ-tocopherol methyltransferase (γ-TMT) is found to be the rate limiting factor in soybean which influences the tocopherol composition. Using Agrobacterium-mediated transformation, we overexpressed the γ-TMT gene of Perilla frutescens under the control of the seed-specific promoter vicillin in cultivar Pusa 16. Transgene integration and expression was confirmed in five independently transformed GUS positive soybean plants by polymerase chain reaction (PCR), Southern hybridization, and reverse transcriptase-PCR (RT-PCR). High-performance liquid chromatography (HPLC) analysis showed that overexpression of Pf-γ-TMT resulted in efficient conversion of γ-tocopherol to α-tocopherol and concomitant increase in seed α-tocopherol content in RT-PCR positive plants. The protocol was successfully applied to three more cultivars PK 416, Gujarat soybean 1, and VL soya 1 in which seeds of transformed plants showed elevated level of α-tocopherol than wild-type seeds.

Agrobacterium tumefaciens-mediated in planta seed transformation strategy in sugarcane.

KEY MESSAGE: An efficient, reproducible and genotype-independent in planta transformation has been standardized for sugarcane using seed as explant. Transgenic sugarcane production through Agrobacterium infection followed by in vitro regeneration is a time-consuming process and highly genotype dependent. To obtain more number of transformed sugarcane plants in a relatively short duration, sugarcane seeds were infected with Agrobacterium tumefaciens EHA 105 harboring pCAMBIA 1304-bar and transformed plants were successfully established without undergoing in vitro regeneration. Various factors affecting sugarcane seed transformation were optimized, including pre-culture duration, acetosyringone concentration, surfactants, co-cultivation, sonication and vacuum infiltration duration. The transformed sugarcane plants were selected against BASTA(®) and screened by GUS and GFP visual assay, PCR and Southern hybridization. Among the different combinations and concentrations tested, when 12-h pre-cultured seeds were sonicated for 10 min and 3 min vacuum infiltered in 100 µM acetosyringone and 0.1 % Silwett L-77 containing Agrobacterium suspension and co-cultivated for 72-h showed highest transformation efficiency. The amenability of the standardized protocol was tested on five genotypes. It was found that all the tested genotypes responded favorably, though CoC671 proved to be the best responding cultivar with 45.4 % transformation efficiency. The developed protocol is cost-effective, efficient and genotype independent without involvement of any tissue culture procedure and can generate a relatively large number of transgenic plants in approximately 2 months.

An investigation on the cytotoxicity and caspase-mediated apoptotic effect of biologically synthesized silver nanoparticles using Podophyllum hexandrum on human cervical carcinoma cells.

Now-a-days synthesis and characterization of silver nanoparticles (AgNPs) through biological entity is quite interesting to employ AgNPs for various biomedical applications in general and treatment of cancer in particular. This paper presents the green synthesis of AgNPs using leaf extract of Podophyllum hexandrum Royle and optimized with various parameters such as pH, temperature, reaction time, volume of extract and metal ion concentration for synthesis of AgNPs. TEM, XRD and FTIR were adopted for characterization. The synthesized nanoparticles were found to be spherical shaped with average size of 14 nm. Effects of AgNPs were analyzed against human cervical carcinoma cells by MTT Assay, quantification of ROS, RT-PCR and western blotting techniques. The overall result indicates that AgNPs can selectively inhibit the cellular mechanism of HeLa by DNA damage and caspase mediated cell death. This biological procedure for synthesis of AgNPs and selective inhibition of cancerous cells gives an alternative avenue to treat human cancer effectively.

A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal.

Withanolide is one of the most extensively exploited steroidal lactones, which are biosynthesized in Withania somnifera. Its production from cell suspension culture was analyzed to defeat limitations coupled with its regular supply from the plant organs. In order to optimize the different factors for sustainable production of withanolides and biomass accumulations, different concentrations of auxins or cytokinins and their combinations, carbon sources, agitation speed, organic additives and seaweed extracts was studied in cell suspension culture. Maximum biomass accumulation (16.72 g fresh weight [FW] and 4.18 g dry weight [DW]) and withanolides production (withanolide A 7.21 mg/g DW, withanolide B 4.23 mg/g DW, withaferin A 3.88 mg/g DW and withanone 6.72 mg/g DW) were achieved in the treatment of Gracilaria edulis extract at 40 % level. Organic additive L-glutamine at 200 mg/l in combination with picloram (1 mg/l) and KN (0.5 mg/l) promoted growth characteristics (11.87 g FW and 2.96 g DW) and withanolides synthesis (withanolide A 5.04 mg/g DW, withanolide B 2.59 mg/g DW, withaferin A 2.36 mg/g DW and withanone 4.32 mg/g DW). Sucrose at 5 % level revolved out to be a superior carbon source yielded highest withanolides production (withanolide A 2.88 mg/g DW, withanolide B 1.48 mg/g DW, withaferin A 1.35 mg/g DW and withanone 2.47 mg/g DW), whereas biomass (7.28 g FW and 1.82 g DW) was gratefully increased at 2 % level of sucrose in cell suspension culture. This optimized protocol can be utilized for large scale cultivation of W. somnifera cells in industrial bioreactors for mass synthesis of major withanolides.

Optimization of carbon source for hairy root growth and withaferin A and withanone production in Withania somnifera.

This study optimized carbon sources in half MS liquid medium for maximum biomass accumulation and withanolides production in hairy root culture of Withania somnifera. The highest production of withaferin A and withanone was achieved when sucrose and sucrose+glucose were used individually as carbon sources. The hairy root suspension culture supplemented with a lower level of sucrose (2%) favored hairy root biomass accumulation (1.41 g DW) followed by sucrose+glucose (2+1) when compared with other carbon sources in half MS liquid medium after 40 days of culture. The hairy roots grown on sucrose (4%) enriched half MS liquid medium stimulated higher production of withaferin A (2.21 mg/g DW) and withanone (2.41 mg/g DW) on the 40th day of culture, followed by sucrose+glucose (4+1%) compared with glucose, fructose, maltose and other combinations tested.

Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal.

Adventitious root cultures derived from leaf derived callus of Withania somnifera (L.) Dunal were treated with methyl jasmonate and salicylic acid independently. Biomass accumulation, culture age, elicitation period, and culture duration were optimized for higher withanolides production in the two best-responding varieties collected from Kolli hills (Eastern Ghats) and Cumbum (Western Ghats) of Tamil Nadu, India. Between the two elicitors, salicylic acid (SA) improved the production of major withanolides (withanolide A, withanolide B, withaferin A, and withanone) as well as minor constituents (12-deoxy withastramonolide, withanoside V, and withanoside IV) in the Kolli hills variety. Treatment of root biomass (11.70 g FW) on 30-day-old adventitious root cultures with 150 µM SA for 4 h elicitor exposure period resulted in the production of 64.65 mg g(-l) dry weight (DW) withanolide A (48-fold), 33.74 mg g(-l) DW withanolide B (29-fold), 17.47 mg g(-l) DW withaferin A (20-fold), 42.88 mg g(-l) DW withanone (37-fold), 5.34 mg g(-l) DW 12-deoxy withastramonolide (nine fold), 7.23 mg g(-l) DW withanoside V (seven fold), and 9.45 mg g(-l) DW withanoside IV (nine fold) after 10 days of elicitation (40th day of culture) when compared to untreated cultures. This is the first report on the use of elicitation strategy on the significant improvement in withanolides production in the adventitious root cultures of W. somnifera.