Metabolic engineering in hairy roots: An outlook on production of plant secondary metabolites.

Plants are one of the vital sources of secondary metabolites. These secondary metabolites have diverse roles in human welfare, including therapeutic implication. Nevertheless, secondary metabolite yields obtained through the exploitation of natural plant populations is insufficient to meet the commercial demand due to their accumulation in low volumes. Besides, in-planta synthesis of these important metabolites is directly linked with the age and growing conditions of the plant. Such limitations have paved the way for the exploration of alternative production methodologies. Hairy root cultures, induced after the interaction of plants with Rhizobium rhizogenes (Agrobacterium rhizogenes), are a practical solution for producing valuable secondary metabolite at low cost and without the influence of seasonal, geographic or climatic variations. Hairy root cultures also offer the opportunity to get combined with other yield enhancements strategies (precursor feeding, elicitation and metabolic engineering) to further stimulate and/or enhance their production potential. Applications of metabolic engineering in exploiting hairy root cultures attracted the interest of several research groups as a means of yield enhancement. Currently, several engineering approaches like overexpression and silencing of pathway genes, and transcription factor overexpression are used to boost metabolite production, along with the contextual success of genome editing. This review attempts to cover metabolic engineering in hairy roots for the production of secondary metabolites, with a primary emphasis on alkaloids, and discusses prospects for taking this research forward to meet desired production demands.

Hydrogen sulfide-mediated mitigation and its integrated signaling crosstalk during salinity stress.

Environmental stresses negatively affect plant development and significantly influence global agricultural productivity. The growth suppression due to soil salinity involves osmotic stress, which is accompanied by ion toxicity, nutritional imbalance, and oxidative stress. The amelioration of salinity stress is one of the fundamental goals to be achieved to ensure food security and better meet the issues related to global hunger. The application of exogenous chemicals is the imperative and efficient choice to alleviate stress in the agricultural field. Among them, hydrogen sulfide (H2 S, a gasotransmitter) is known for its efficient role in stress mitigation, including salinity stress, along with other biological features related to growth and development in plants. H2 S plays a role in improving photosynthesis and ROS homeostasis, and interacts with other signaling components in a cascade fashion. The current review gives a comprehensive view of the participation of H2 S in salinity stress alleviation in plants. Further, its crosstalk with other stress ameliorating signaling component or supplement (e.g., NO, H2 O2 , melatonin) is also covered and discussed. Finally, we discuss the possible prospects to meet with success in agricultural fields.

3D Bioprinting in Plant Science: An Interdisciplinary Approach.

Here we highlight advances and opportunities for using 3D bioprinting in plant biology research that could lead to low-cost solutions for biomedical and other applications. For example, the development of plant cell-based and plant-inspired 3D-printed constructs could provide information about single-cell, tissue, and whole-plant interactions with the surrounding environment.

Artificial neural network-based model for the prediction of optimal growth and culture conditions for maximum biomass accumulation in multiple shoot cultures of Centella asiatica.

An artificial neural network (ANN)-based modelling approach is used to determine the synergistic effect of five major components of growth medium (Mg, Cu, Zn, nitrate and sucrose) on improved in vitro biomass yield in multiple shoot cultures of Centella asiatica. The back propagation neural network (BPNN) was employed to predict optimal biomass accumulation in terms of growth index over a defined culture duration of 35 days. The four variable concentrations of five media components, i.e. MgSO4 (0, 0.75, 1.5, 3.0 mM), ZnSO4 (0, 15, 30, 60 µM), CuSO4 (0, 0.05, 0.1, 0.2 µM), NO3 (20, 30, 40, 60 mM) and sucrose (1, 3, 5, 7 %, w/v) were taken as inputs for the ANN model. The designed model was evaluated by performing three different sets of validation experiments that indicated a greater similarity between the target and predicted dataset. The results of the modelling experiment suggested that 1.5 mM Mg, 30 µM Zn, 0.1 µM Cu, 40 mM NO3 and 6 % (w/v) sucrose were the respective optimal concentrations of the tested medium components for achieving maximum growth index of 1654.46 with high centelloside yield (62.37 mg DW/culture) in the cultured multiple shoots. This study can facilitate the generation of higher biomass of uniform, clean, good quality C. asiatica herb that can efficiently be utilized by pharmaceutical industries.

Scale-Up of Agrobacterium rhizogenes-Mediated Hairy Root Cultures of Rauwolfia serpentina: A Persuasive Approach for Stable Reserpine Production.

Roots of Rauwolfia serpentina, also known as "Sarpagandha" possess high pharmaceutical value due to the presence of reserpine and other medicinally important terpene indole alkaloids. Ever increasing commercial demand of R. serpentina roots is the major reason behind the unsystematic harvesting and fast decline of the species from its natural environment. Considering Agrobacterium rhizogenes-mediated hairy root cultures as an alternative source for the production of plant-based secondary metabolites, the present optimized protocol offers a commercially feasible method for the production of reserpine, the most potent alkaloid from R. serpentina roots. This end-to-end protocol presents the establishment of hairy root culture from the leaf explants of R. serpentina through the infection of A. rhizogenes strain A4 in liquid B5 culture medium and its up-scaling in a 5 L bench top, mechanically agitated bioreactor. The transformed nature of roots was confirmed through PCR-based rol A gene amplification in genomic DNA of putative hairy roots. The extraction and quantification of reserpine in bioreactor grown roots has been done using monolithic reverse phase high-performance liquid chromatography (HPLC).

Hairy root biotechnology--indicative timeline to understand missing links and future outlook.

Agrobacterium rhizogenes-mediated hairy roots (HR) were developed in the laboratory to mimic the natural phenomenon of bacterial gene transfer and occurrence of disease syndrome. The timeline analysis revealed that during 90 s, the research expanded to the hairy root-based secondary metabolite production and different yield enhancement strategies like media optimization, up-scaling, metabolic engineering etc. An outlook indicates that much emphasis has been given to the strategies that are helpful in making this technology more practical in terms of high productivity at low cost. However, a sequential analysis of literature shows that this technique is upgraded to a biotechnology platform where different intra- and interdisciplinary work areas were established, progressed, and diverged to provide scientific benefits of various hairy root-based applications like phytoremediation, molecular farming, biotransformation, etc. In the present scenario, this biotechnology research platform includes (a) elemental research like hairy root-mediated secondary metabolite production coupled with productivity enhancement strategies and (b) HR-based functional research. The latter comprised of hairy root-based applied aspects such as generation of agro-economical traits in plants, production of high value as well as less hazardous molecules through biotransformation/farming and remediation, respectively. This review presents an indicative timeline portrayal of hairy root research reflected by a chronology of research outputs. The timeline also reveals a progressive trend in the state-of-art global advances in hairy root biotechnology. Furthermore, the review also discusses ideas to explore missing links and to deal with the challenges in future progression and prospects of research in all related fields of this important area of plant biotechnology.

Hairy root biotechnology of Rauwolfia serpentina: a potent approach for the production of pharmaceutically important terpenoid indole alkaloids.

Hairy root cultures of Rauwolfia serpentina induced by Agrobacterium rhizogenes have been investigated extensively for the production of terpenoid indole alkaloids. Various biotechnological developments, such as scaling up in bioreactors, pathway engineering etc., have been explored to improve their metabolite production potential. These hairy roots are competent for regenerating into complete plants and show survival and unaltered biosynthetic potential during storage at low temperature. This review provides a comprehensive account of the hairy root cultures of R. serpentina, their biosynthetic potential and various biotechnological methods used to explore the production of pharmaceutically important terpenoid indole alkaloids. The review also indicates how biotechnological endeavors might improve the future progress of research for production of alkaloids using Rauwolfia hairy roots.

Plant regeneration, genetic fidelity, and active ingredient content of encapsulated hairy roots of Picrorhiza kurrooa Royle ex Benth.

Among five hairy root lines of Picrorhiza kurrooa that were established through Agrobacterium rhizogenes, one (H7) was selected for encapsulation due to high accumulation of picrotin and picrotoxinin (8.3 and 47.6 µg/g DW, respectively). Re-grown encapsulated roots induced adventitious shoots with 73 % frequency on MS medium supplemented with 0.1 µM 6-benzylaminopurine, following 6 months of storage at 25 °C. Regenerated plantlets had 85 % survival after 2 months. Regenerants were of similar morphotype having increased leaf number and branched root system as compared to non-transformed plants. The transformed nature of the plants was confirmed through PCR and Southern blot analysis. Genetic fidelity analysis of transformed plants using RAPD and ISSR showed 5.2 and 3.6 % polymorphism, respectively. Phytochemical analysis also showed that picrotin and picrotoxinin content were similar in hairy root line and its regenerants.

Efficiency of neural network-based combinatorial model predicting optimal culture conditions for maximum biomass yields in hairy root cultures.

KEY MESSAGE : ANN-based combinatorial model is proposed and its efficiency is assessed for the prediction of optimal culture conditions to achieve maximum productivity in a bioprocess in terms of high biomass. A neural network approach is utilized in combination with Hidden Markov concept to assess the optimal values of different environmental factors that result in maximum biomass productivity of cultured tissues after definite culture duration. Five hidden Markov models (HMMs) were derived for five test culture conditions, i.e. pH of liquid growth medium, volume of medium per culture vessel, sucrose concentration (%w/v) in growth medium, nitrate concentration (g/l) in the medium and finally the density of initial inoculum (g fresh weight) per culture vessel and their corresponding fresh weight biomass. The artificial neural network (ANN) model was represented as the function of these five Markov models, and the overall simulation of fresh weight biomass was done with this combinatorial ANN-HMM. The empirical results of Rauwolfia serpentina hairy roots were taken as model and compared with simulated results obtained from pure ANN and ANN-HMMs. The stochastic testing and Cronbach's α-value of pure and combinatorial model revealed more internal consistency and skewed character (0.4635) in histogram of ANN-HMM compared to pure ANN (0.3804). The simulated results for optimal conditions of maximum fresh weight production obtained from ANN-HMM and ANN model closely resemble the experimentally optimized culture conditions based on which highest fresh weight was obtained. However, only 2.99 % deviation from the experimental values could be observed in the values obtained from combinatorial model when compared to the pure ANN model (5.44 %). This comparison showed 45 % better potential of combinatorial model for the prediction of optimal culture conditions for the best growth of hairy root cultures.

ISSR and RAPD based evaluation of genetic stability of encapsulated micro shoots of Glycyrrhiza glabra following 6 months of storage.

In vitro grown axillary micro shoots of Glycyrrhiza glabra were encapsulated in alginate beads. Following 6 months of normal storage at 25 ± 2°C the re growth of encapsulated G. glabra micro shoots, reached 98% within 30 days of incubation on MS medium supplemented with 0.1 mg/l IAA. Re growth was characterized by the development of both shoot and root from single encapsulated micro shoot. Healthy plants were established to glass house with 95% survival. The genetic fidelity of plants obtained after conversion of alginate beads was ascertained through 10 RAPD and 13 ISSR primers. Of the 10 RAPD primers tested, 6 of them produced 14 clear and reproducible amplicons with an average of 2.3 bands per primer out of which 28.57% were polymorphic generated by only two primers. Eight ISSR primers produced total 37 bands ranging between 300 and 3,500 bp length. Number of scorable bands for each primer varied from 3 to 8 with an average of 4.6 bands per primer. Cluster analysis from ISSR and RAPD showed that all the tested plants including the mother plant distributed in two major groups with similarity coefficient ranging from 0.91 to 0.96 for RAPD and 0.89 to 0.97 for ISSR.

Conservation and multiplication of encapsulated micro shoots of Rauvolfia vomitoria--an endangered medicinal tree: ISSR and RAPD based evaluation of genetic fidelity of converted plantlets.

The in vitro grown axillary micro shoots of Rauvolfia vomitoria were encapsulated in alginate beads. Following 6 months of normal storage at 25 +/- 2 degrees C the regrowth of encapsulated micro shoots, reached 95.2% within 40 days of incubation on MS medium containing 1.0 mg/L BAP and 0.1 mg/L NAA. Among the responding encapsulated explants 69.6% showed emergence of multiple shoots. The developing shoots showed rhizogenesis in two weeks following their transfer to rooting medium. Healthy plants were established in a glass house with 95% survival. Of the 50 RAPD primers tested, 10 produced 23 clear and reproducible amplicons, with an average of 2.3 bands per primer. Eleven ISSR primers produced a total of 42 bands, with a size range of 0.1-1.9 kb. The number of scorable bands for each primer varied from 2 to 6, with an average of 3.81. The similarity matrix, calculated individually from the results obtained from ISSR and RAPD analysis, showed similarity coefficients ranging from 1.0 for RAPD and 0.85 to 1.0 for ISSR.

Elicitor-induced cellular and molecular events are responsible for productivity enhancement in hairy root cultures: an insight study.

A wide range of external stress stimuli triggers a plant cell to undergo a complex network of reactions that ultimately lead to the synthesis and accumulation of secondary metabolites. These secondary metabolites help the plant to survive under stress challenge. The potential of biotic and abiotic elicitors for the induction and enhancement of secondary metabolite production in various culture systems including hairy root (HR) cultures is well-known. The elicitor-induced defense responses involves signal perception of elicitor by a cell surface receptor followed by its transduction involving some major cellular and molecular events including activation of major secondary message signaling pathways. This result in induction of gene expressions escorting to the synthesis of various proteins mainly associated with plant defense responses and secondary metabolite synthesis and accumulation. The review discusses the elicitor-induced various cellular and molecular events and correlates them with enhanced secondary metabolite synthesis in HR systems. Further, this review also concludes that combining elicitation with in-silico approaches enhances the usefulness of this practice in better understanding and identifying the rate-limiting steps of biosynthetic pathways existing in HRs which in turn can contribute towards better productivity by utilizing metabolic engineering aspects.

An extensive case study of hairy-root cultures for enhanced secondary-metabolite production through metabolic-pathway engineering.

An intrinsic improvement is taking place in the methodologies for the development of culture systems with first-rate production of plant-based molecules. The blending of HR (hairy root) cultures with ME (metabolic engineering) approaches offers new insights into, and possibilities for, improving the system productivity for known and/or novel high-value plant-derived active compounds. The introduction and expression of foreign genes in plants results in improvement of cellular activities by manipulating enzymatic, regulatory and transport function of the cell. The rational amendments in the rate-limiting steps of a biosynthetic pathway as well as inactivating the inefficient pathway(s) for by-product formation can be accomplished either through single-step engineering or through the multi-step engineering. The hierarchical control of any metabolic process can lead the engineer to apply the ME ideas and principles to any of the strata, including transcriptional, moving on to translational and enzymatic activity. The HR culture systems offer a remarkable potential for commercial production of a number of low-volume, but high-value, secondary metabolites. Taking HR as a model system, in the present review, we discuss engineering principles and perceptions to exploit secondary-metabolite pathways for the production of important bioactive compounds. We also talk about requisites and possible challenges that occur during ME, with emphasis on examples of various HR systems. Furthermore, it also highlights the utilization of global information obtained from '-omic' platforms in order to explore pathway architecture, structural and functional aspects of important enzymes and genes that can support the design of sets of engineering, resulting in the generation of wide-ranging views of DNA sequence-to-metabolite passageway networking and their control to obtain desired results.

A neural network approach for the prediction of in vitro culture parameters for maximum biomass yields in hairy root cultures.

The present study deals with ANN based prediction of culture parameters in terms of inoculum density, pH and volume of growth medium per culture vessel and sucrose content of the growth medium for Glycyrrhiza hairy root cultures. This kind of study could be a model system in exploitation of hairy root cultures for commercial production of pharmaceutical compounds using large bioreactors. The study is aimed to evaluate the efficiency of regression neural network and back propagation neural network for the prediction of optimal culture conditions for maximum hairy root biomass yield. The training data for regression and back propagation networks were primed on the basis of function approximation, where final biomass fresh weight (f(wt)) was considered as a function of culture parameters. On this basis the variables in culture conditions were described in the form of equations which are for inoculum density: y=0.02x+0.04, for pH of growth medium: y=x+2.8, for sucrose content in medium: y=9.9464x+(-9.7143) and for culture medium per culture vessel: y=10x. The fresh weight values obtained from training data were considered as target values and further compared with predicted fresh weight values. The empirical data were used as testing data and further compared with values predicted from trained networks. Standard MATLAB inbuilt generalized regression network with radial basis function radbas as transfer function in layer one and purelin in layer two and back propagation having purelin as transfer function in output layer and logsig in hidden layer were used. Although in comparative assessment both the networks were found efficient for prediction of optimal culture conditions for high biomass production, more accuracy in results was seen with regression network.

Genetic transformation studies and scale up of hairy root culture of Glycyrrhiza glabra in bioreactor

The study was undertaken to induce hairy roots in Glycyrrhiza glabra in leaf explants and to optimize the nutritional requirement for its growth kinetics at shake flask and bioreactor level. Pathogenecity of Agrobacterium depends upon transformation ability of strain and age, type, and physiological state of explants. Agrobacterium rhizogenes strain K599 was used to infect leaf explants of G. glabra. Explants of different age groups were obtained from 2 to 5 weeks old in vitro grown cultures. Bacterial strain K599 could induce hairy roots in 3 and 4 weeks old leaf explants cultured on B5, MS, NB and WP basal semi-solid medium. Leaf explants of 2 and 5 weeks old culture were not responsive to bacterial infection in terms of hairy root induction. Maximum transformation frequency (TF) of tested bacterial strain was 47 percent obtained in 3 weeks old explants after 25 days of incubation on MS basal semi solid medium. NB and B5 both media composition showed 20 percent of transformation frequency after 28 and 38 days respectively. WP medium did not support induction of roots in cultured leaf explants infected with A. rhizogenes strain K599even after 50 days of incubation. Further, when all the four media combinations were tested for root growth it was found that though WP was not responsive for hairy root induction, yet all four basal media supported hairy root growth and a gradual increase in fresh weight biomass was observed with an increase in culture duration. However amongst all, the NB medium composition supported best growth of hairy roots followed by MS, B5 and WP media. About 20 times increase in root biomass on fresh weight basis was recorded after 45days of culture in NB medium. Initial inoculum of roots (0.18 g. F.wt./ flask) containing 50 ml of liquid culture medium produced 3.59 g (F. wt.) biomass. A fast growing hairy root clone G6 was grown in a 5 l capacity mechanically agitated bioreactor provided with a nylon mesh septum. After 30 days of sterile...