In vitro production of doubled haploid plants in Camellia spp. and assessment of homozygosity using microsatellite markers.

In this report, in vitro doubled haploid (DH) plants were established in two tea (Camellia spp) cultivars, TV21 (Assam Type) and TV19 (Cambod Type). Androgenic globular stage haploid embryos, obtained via callusing from microspores at an early-to-late uninucleate stage in anther cultures, were diploidized by colchicine treatments at varying concentrations and durations under dark incubation at 25 ± 2 °C temperature. Thereafter, treated embryos were transferred to development medium, Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP; 1 µM) + gibberellic acid (GA3; 0.3 µM) + L-glutamine (80 mg l-1) + L-serine (20 mg l-1) and incubated in diffused light. Ploidy of germinating embryos was evaluated by flow-cytometry and cytological squash preparation. High chromosome doubling, 76.89% and 67.34%, was obtained in embryos of TV21 and TV19, respectively, at 0.2% colchicine treatment for 24 h. The DH plants were further multiplied via axillary-bud proliferation on multiplication medium, MS + glucose (30 g l-1) + BAP (5 µM) + GA3 (0.5 µM) + IBA (0.5 µM) + L- glutamine (80 mg l-1) + L-serine (20 mg l-1). Rooting of shoots was achieved on ⅓ MS basal medium within 50 days of inoculation when shoots were pre-treated with IBA (175 µM) for ten days. The rooted plants were acclimatized in field. Homozygosity in diploidized plants was validated by SSR marker.

An interdisciplinary approach towards sustainable and higher steviol glycoside production from in vitro cultures of Stevia rebaudiana.

Stevia rebaudiana is one of the vastly acclaimed commercial plant in the world and belongs to Asteraceae family. The exclusive advantage of Stevia over artificial sweeteners is impeccable and targets its potentiality to the presence of diterpene glycosides. Moreover, the flaunting sweetness of steviol glycosides with associated medicinal benefits, turns the plant to be one of the most economic assets, globally. As compared to vegetative propagation through stem-cuttings, plant tissue culture is the most suitable approach in obtaining true-to-type plants of superior quality. During last few decades, significant in vitro propagation methods have been developed and still the research is ongoing. The present review discusses the tissue culture perspectives of S. rebaudiana, primarily focusing on the mineral nutrition, growth regulators and other accessory factors, motioning the optimum growth and development of the plant. Another crucial aspect is the generation of sweeter varieties in order to reduce the bitter-off taste, which is noticed after the consumption of the leaves. The in vitro cultures pose an efficient alternative system for production of steviol glycosides, with higher rebaudioside(s) content. Moreover, the review also covers the recent approaches pertaining to scale-up studies and genome editing perspectives.

Chromium tolerance, bioaccumulation and localization in plants: An overview.

In the current industrial scenario, chromium (Cr) as a metal is of great importance, but poses a major threat to the environment. Phytoremediation provides an environmentally sustainable, ecofriendly, cost effective approach for environmental cleanup of Cr. This review presents the current status of phytoremediation research with particular emphasis on cleanup of Cr contaminated soil and water systems. It gives a detailed account of the work done by different authors on the Cr bioavailability, uptake pathway, toxicity and storage in plants following the phytoextraction mechanism. This paper also describes recent findings related to Cr localization in hyperaccumulator plants. It gives an insight into the processes and mechanisms that allow plants to remove Cr from contaminated sites under varying conditions. These detailed knowledge of changes in plant metabolic pool in response to Cr stress would immensely help understand and improve the phytoextraction process. Further, this review provides a detailed understanding of Cr uptake and detoxification mechanism by plants that can be applied in developing a suitable approach for a better applicability of the process.

Kinetics, biochemical and factorial analysis of chromium uptake in a multi-ion system by Tradescantia pallida (Rose) D. R. Hunt.

Discharge of wastewater from electroplating and leather industries is a major concern for the environment due to the presence of toxic Cr6+ and other ions, such as sulfate, nitrate, phosphate, etc. This study evaluated the potential of Tradescantia pallida, a plant species known for its Cr bioaccumulation, for the simultaneous removal of Cr6+, SO42-, NO3-, and PO43-. The effect of different co-ions on Cr6+ removal by T. pallida was examined following the Plackett-Burman design of experiments carried out under batch hydroponics conditions. The results revealed a maximum removal of 84% Cr6+, 87% SO42-, 94% NO3- and 100% PO43- without any phytotoxic effect on the plant for an initial Cr6+ concentration in the range 5-20 mg L-1. SO42- and NO3- enhanced Cr uptake at a high initial Cr concentration (20 mg L-1), whereas PO43- did not affect Cr uptake both at high and low initial Cr concentrations. The Cr6+ removal kinetics in the presence of different ions was well described by the pseudo-second-order kinetic model which revealed that both biosorption and bioaccumulation of the metal played an important role in Cr6+ removal. Increase in the total carbohydrate and protein content of the plant following Cr6+ and co-ions exposure indicated a good tolerance of the plant toward Cr6+ toxicity. Furthermore, enhancement in the lipid peroxidation and catalase activity in T. pallida upon Cr6+ exposure revealed a maximum stress-induced condition in the plant. Overall, this study demonstrated a very good potential of the plant T. pallida for Cr6+ removal from wastewater even in the presence of co-ions.

Isolation and quantification of antimalarial N-alkylamides from flower-head derived in vitro callus cultures of Spilanthes paniculata.

This is the pioneer work reporting on simple procedure for synchronized determination and quantification of two biologically active N-alkylamides, (2E,6Z,8E)-N-isobutyl-2,6,8-decatrienamide (spilanthol) and (2E,4Z)-N-isobutyl-2,4-undecadiene-8,10-diynamide (UDA), using in vitro callus cultures from flower-heads of Spilanthes paniculata. The extracts were purified using preparative thin layer chromatography (TLC) and finest separation of compounds was optimized using high performance liquid chromatography (HPLC). Eventually, N-alkylamides were validated by mass spectrometry. Linearity curve with its regression coefficients (R2) obtained for both these alkylamides was 0.99. While spilanthol was quantified using tentative standard dodeca-2(E),4(E)-dienoic acid due to the non-availability of commercial standard and the precision of a developed method was evaluated in terms of relative standard deviation by measuring inter- and intra-days variation 3.52% and 1.74%, respectively. Similarly, calibration curve was obtained for the compound UDA isolated from flower-head explants from field grown parental plant with its inter- and intra-day RSD values as 4.33% and 3.61%, respectively. With this protocol, a very high yield of 2.23 mg/g of spilanthol and 4.30 mg/g dry weight (DW) of UDA, was obtained, simultaneously, from callus cultures. Flower-heads from parent plants, used as control, showed negligible amount of spilanthol and quantity of UDA was marginally higher than that in callus cultures. The highly stable biotherapeutic spilanthol and UDA with m/z 222 and m/z 230, respectively, showed retardation of malaria parasite development through blockage at ring stage of erythrocytic schizogony and ultimately lead to parasite death. The effect on parasite was additive. This study signifies the utility of in vitro cell cultures for therapeutic compound production, throughout the year, at higher yield for down-stream applications.

Evaluation of Cr(VI) Exposed and Unexposed Plant Parts of Tradescantia pallida (Rose) D. R. Hunt. for Cr Removal from Wastewater by Biosorption.

Phytoremediation is an efficient method for the removal of heavy metals from contaminated systems. A productive disposal of metal accumulating plants is a major concern in current scenario. In this work, Cr(VI) accumulating Tradescantia pallida plant parts were investigated for its reuse as a biosorbent for the removal of Cr(VI) ions. The effect of pH, contact time, sorbent dosage, Cr(VI) concentration and temperature was examined to optimize these process parameters. Results showed that Cr(VI) exposed/unexposed T. pallida leaf biomass could remove 94% of chromium with a sorption capacity of 64.672 mg g(-1). Whereas the kinetics of Cr(VI) biosorption was well explained by the pseudo second-order kinetic model, the Langmuir model better described the data on Cr(VI) sorption isotherm compared with the Freundlich model. The changes in the free energy (ΔG°), entropy (ΔS°) and enthalpy (ΔH°) were found to be -5.276 kJ mol(-1), 0.391 kJ mol(-1) K(-1) and 11.346 kJ mol(-1), respectively, which indicated the process to be spontaneous, feasible and endothermic in nature. FTIR spectra of T. pallida leaf biomass revealed the active participation of ligands, such as -NH, amide, hydroxyl and sulphonate groups present in the biomass for Cr(VI) binding, SEM analysis revealed a porous structure of the biosorbent for an easy uptake of Cr(VI).

Chromium(VI) accumulation and tolerance by Tradescantia pallida: biochemical and antioxidant study.

Tradescantia pallida (Wandering jew)-a succulent perennial herb-was screened to be a potent chromium (Cr) accumulator. Its ability to grow under Cr stress was examined by studying biochemical changes and physiological response of the plant in presence of 5-20 mg L(-1) Cr(VI) concentration in hydroponic environment for up to ca. 90 days. Average Cr(VI) bioaccumulation in plant roots reached about 408 µg g(-1) dry weight (dw) after 30 days and up to 536 µg g(-1)dw after 60 days of culture. Biochemical changes in the plant exposed to Cr(VI) indicated a reduction in the total carbohydrate and protein content. Furthermore, lipid peroxidation, catalase, peroxidase and ascorbate peroxidase activity were measured in different parts of the plant exposed to Cr(VI). Increased activities of these enzymes showed their important role in overcoming the Cr-induced oxidative stress on the plant.

Evaluation of nutrient uptake and physical parameters on cell biomass growth and production of spilanthol in suspension cultures of Spilanthes acmella Murr.

Spilanthes acmella Murr. has a plethora of highly valuable biologically active compounds and has been listed as one of the important medicinal plants of the world. However, no perceptible biotechnological advances have been made for this genus to exploit or enhance its utility. To nullify the effect of seasonal variations, the present report is the first attempt to establish in vitro cell suspension cultures and to evaluate the production of spilanthol from them, a key component of the plant responsible for most of its pharmaceutical activities. The study examined the biomass growth in relation to the consumption of major nutrients and sucrose, agitation speed and dynamic change in pH. Results indicated that the consumption of phosphate resulted in the onset of decline phase in cultures. Spilanthol production was observed to be growth associated and maximum production occurred on the 15th day. Among the carbon sources, the highest production of spilanthol as 91.4 µg g(-1) DW was recorded in the medium supplemented with sucrose, followed by glucose which produced 56.8 µg g(-1) DW of spilanthol. Spilanthol could not be detected in fructose containing medium. Maximum viable cultures were obtained at a rotation speed of cells at 120 rpm. This study signifies the utility of Spilanthes suspension cultures for biosynthesis and constant production of spilanthol, throughout the year. The results of present study are useful for further scale-up process.

Increased production of azadirachtin from an improved method of androgenic cultures of a medicinal tree Azadirachta indica A. Juss.

Present report is the first direct evidence of azadirachtin production in androgenic haploid cultures of Azadirachta indica, a woody medicinal tree. Anther cultures at early-late-uninucleate stage of microspores were established on MS medium with BAP (5 µM), 2,4-D (1 µM) and NAA (1 µM) containing 12% sucrose. The calli, induced, were further multiplied on 2,4-D and Kinetin media. Shoots, differentiated on BAP (2.2 µM) + NAA (0.05 µM) medium, were elongated on MS + BAP (0.5 µM) and multiplied on MS + BAP (1 µM) + CH (250 mg/l). Thereafter, the shoots were rooted on » MS + IBA (0.5 µM). Cytological analysis of the calli and regenerants have confirmed their haploid status with the chromosome number as 2n = x = 12. The haploid cell lines and leaves from in vitro grown plantlets were analyzed for azadirachtin by RP-HPLC and mass spectroscopy. Maximum azadirachtin (728.41 µg/g DW) was detected in calli supporting best shoot proliferation while least (49 µg/g DW) was observed in an undifferentiated line from maintenance medium. This study has brought us a step closer to develop genetically pure lines that could serve as new and attractive alternative ways of homogeneous controlled production of high value compounds, round the year, independent of geographical and climatic barrier.

Effect of culture conditions on synthesis of triterpenoids in suspension cultures of Lantana camara L.

Present report is aimed to study the batch kinetics of Lantana camara. Dynamic changes of parameters, such as pH, conductivity, wet and dry cell concentrations, consumption of major nutrients, carbon source and agitation speeds were investigated to understand the culture characteristics of suspended cells grown on MS + BAP + 2,4-D + NAA in shake flasks. Results indicated that the consumption of phosphate resulted in the onset of stationary phase in cultures. Maltose as carbon source resulted in production of maximum triterpenoid content (31.08 mg/L) while the least was found on glucose (10.69 mg/L). Notably, both did not support accumulation of betulinic acid. Sucrose, although stood second in terms of quantity (21.6 mg/L), supported the production of all the three triterpenoids-oleanolic, ursolic and betulinic acids. Maximum viable cultures were obtained at a rotation speed of 120 rpm. The present finding will form a background for further scale-up related studies.

In vitro androgenesis in tree species: an update and prospect for further research.

Most, if not all, trees are outbreeding, highly heterozygous and undergo a long developmental period before reaching their reproductive stage. Classical breeding and cross-pollinating procedures are both unpredictable and time-consuming. In vitro androgenesis is, thus, the most prolific and desirable approach of haploid production. But various attempts to induce androgenic potential in the trees have met with rather limited success, as they ought to be extremely recalcitrant in culture. The success rate in this case is nowhere close to that achieved for some model species like Brassica and Nicotiana. Our review article intends to focus on the overview of androgenic process and all the major contributions till date on tree species with regard to this aspect. We wish to bring together in one place all the important variables used by different workers, that influence androgenic potential immensely like, stage of anther or microspore at culture, media composition, combinations and concentrations of growth regulators, and additives. This will prove to be a worthy guide to all the prospective workers in this area and in designing their experiments further.

An efficient protocol for the production of triploid plants from endosperm callus of neem, Azadirachta indica A. Juss.

Triploid plants of neem were obtained by immature endosperm culture. Immature seeds, at the early dicotyledonous stage of embryo development, is the best explant to raise endosperm callus on MS + NAA (5 mumol/L) + BAP (2 mumol/L) + CH (500 mg L-1). Maximum shoot bud differentiation from the endosperm callus occurred on MS + 5 mumol/L BAP. Shoots were multiplied by forced axillary branching and rooted in vitro. The plants were established in soil. Over 66% of the plants were triploid with chromosome number 2n = 3x = 36. A characteristic feature of the shoots of endosperm origin is the presence of a large number of multi-cellular glands.