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Nanotechnology in Plant Metabolite Improvement and in Animal Welfare

Plant tissue culture plays an important role in plant biotechnology due to its potential for massive production of improved crop varieties and high yield of important secondary metabolites. Several efforts have been made to ameliorate the effectiveness and production of plant tissue culture, using biotic and abiotic factors. Nowadays, the addition of nanoparticles as elicitors has, for instance, gained worldwide interest because of its success in microbial decontamination and enhancement of secondary metabolites. Nanoparticles are entities in the nanometric dimension range: they possess unique physicochemical properties. Among all nanoparticles, silver-nanoparticles (AgNPs) are well-known for their antimicrobial and hormetic effects, which in appropriate doses, led to the improvement of plant biomass as well as secondary metabolite accumulation. This review is focused on the evaluation of the integration of nanotechnology with plant tissue culture. The highlight is especially conveyed on secondary metabolite enhancement, effects on plant growth and biomass accumulation as well as their possible mechanism of action. In addition, some perspectives of the use of nanomaterials as potential therapeutic agents are also discussed. Thus, the information provided will be a good tool for future research in plant improvement and the large-scale production of important secondary metabolites. Elicitation of silver-nanoparticles, as well as nanomaterials, function as therapeutic agents for animal well-being is expected to play a major role in the process. However, nanosized supramolecular aggregates have received an increased resonance also in other fields of application such as animal welfare. Therefore, the concluding section of this contribution is dedicated to the description and possible potential and usage of different nanoparticles that have been the object of work and expertise also in our laboratories.

Aklimatisasi Anggrek Species Hasil Kultur Jaringan Melalui Pemberdayaan Masyarakat Dusun Gempol

Abstract Gempol Village is a village on Mount Ungaran that has made efforts to preserve orchid species. Through mentoring and training from the UNNES Research Team which began in 2011, as well as greenhouse facilitation from PT Indonesia Power, the Gempol village community who are members of the Omah Sawah Community began to make efforts to conserve orchid species. The results of the identification of the problems experienced by community groups as foster partners can be grouped into three aspects, namely knowledge and skills of acclimatization of orchids from plant tissue culture, post-acclimatization management/care, and supporting infrastructure for acclimatization of tissue cultured orchids, where these three aspects are interrelated. The methods used include lecture and question and answer activities, practice, and mentoring. The result achieved is that the orchid species acclimatization activity in Gempol Hamlet, Ngesrepbalong Village has been carried out with satisfactory results. Activities are carried out through training and assistance to community groups who are members of Omah Sawah. The results of the evaluation of the participants showed that the participants' understanding and skills improved after this activity was carried out, even providing ideas for participants to apply to cultivated orchids. The result is enough to generate economic income for the participants, because some of their cultivated orchids are sold. Abstrak Dusun Gempol adalah satu dusun di  Gunung Ungaran yang telah melakukan upaya pelestarian anggrek species hutan . Melalui pendampingan dan pelatihan dari Tim Peneliti UNNES yang dimulai pada tahun 201, serta fasilitasi greenhouse dari PT Indonesia Power, masyarakat Dusun Gempol yang tergabung dalam Komunitas Omah Sawah mulai melakukan upaya pelestarian anggrek species. Hasil identifikasi terhadap permasalahan yang dialami kelompok masyarakat sebagai mitra binaan dapat dikelompokkan menjadi tiga aspek, yaitu pengetahuan dan keterampilan aklimatisasi anggrek hasil kultur jaringan tanaman, pengelolaan/ perawatan pasca aklimatisasi, dan sarana prasarana penunjang aklimatisasi anggrek hasil kutur jaringan, dimana ke tiga aspek ini salingterkait. Metode yang dilakukan, meliputi kegiatan ceramah dan tanya jawab, praktik, dan pendampingan. Hasil yang dicapai adalah kegiatan aklimatisasi anggrek species di Dusun Gempol Desa Ngesrepbalong telah dilaksanakan dengan hasil yang memuaskan. Kegiatan dilaksanakan melalui pelatihan dan pendampingan terhadap kelompok masyarakat yang tergabung di Omah Sawah. Hasil evaluasi terhadap peserta menunjukkan bahwa pemahaman dan ketrampilan peserta meningkat setelah dilakukan kegiatan ini, bahkan memberikan ide bagi peserta untuk menerapkan pada anggrek budidaya. Hasilnya cukup membuahkan pemasukan ekonomi bagi peserta, karena beberapa anggrek budidaya mereka laku dijual.   

Effect of LED Lighting on Physical Environment and Microenvironment on In Vitro Plant Growth and Morphogenesis: The Need to Standardize Lighting Conditions and Their Description

In the last decades, lighting installations in plant tissue culture have generally been renewed or designed based on LED technology. Thanks to this, many different light quality advances are available but, with their massive implementation, the same issue is occurring as in the 1960s with the appearance of the Grolux (Sylvania) fluorescent tubes: there is a lack of a methodological standardization of lighting. This review analyzes the main parameters and variables that must be taken into account in the design of LED-based systems, and how these need to be described and quantified in order to homogenize and standardize the experimental conditions to obtain reproducible and comparable results and conclusions. We have designed an experimental system in which the values of the physical environment and microenvironment conditions and the behavior of plant tissue cultures maintained in cabins illuminated with two lighting designs can be compared. Grolux tubes are compared with a combination of monochromatic LED lamps calibrated to provide a spectral emission, and light irradiance values similar to those generated by the previous discharge lamps, achieving in both cases wide uniformity of radiation conditions on the shelves of the culture cabins. This study can help to understand whether it is possible to use LEDs as one standard lighting source in plant tissue culture without affecting the development of the cultures maintained with the previously regulated protocols in the different laboratories. Finally, the results presented from this caparison indicate how temperature is one of the main factors that is affected by the chosen light source.

Plant Tissue Culture Techniques for Conservation of Biodiversity of Some Plants Appropriate for Propgation in Degraded and Temperate Areas

Integration of nanotechnology in plant tissue culture.

: In the field of plant biology, tissue culture is having colossal applications, for example, the production of disease-free plants and their mass multiplication, germplasm preservation, genetic manipulation to get improved variety as well as the production of biologically active compounds. The integration of nanotechnology and application of nanoparticles (NPs) has shown a positive response in the elimination of microbial contaminants and induction of callus, somatic embryogenesis, organogenesis, production of secondary metabolites, and genetic transformation. This paper aims to highlight some of the recent advancements that came possible through the implementation of nanotechnology in the field of plant tissue culture and also discusses both positives and negatives aspects associated with NPs in plant tissue culture. The prospects through the involvement of recent innovations of nanotechnology such as dendrimers, quantum dots, and carbon nanotubes are also proposed.

Development of Vivorium, a new indoor horticultural ornamental plants via plant tissue culture techniques

Optimisation of plant tissue culture conditions in a popular semi-dwarf indica rice cultivar adt 39 for effective agrobacterium-mediated transformation, impact of chitosan on plant tissue culture: recent applications, plant tissue culture and ultra high diluted studies: suggesting a novel model using in vitro techniques.

Plant tissue culture techniques have been used to evaluate the effects of many different substances and/ or conditions in plant growth and development. It provides information of great value about problems related to basic and applied aspects of plant as well as contributed to understanding of factors responsible for growth, metabolism, synthesis of secondary compounds, stress response. Considering all this wide range of applications and as all plant tissue culture techniques are undergone under axenic and controlled conditions (culture medium composition, light and temperature, for instance), it seems to be a value model for Ultra High Diluted (UHD) studies. Lippia alba is a Brazilian plant that tissue cultures protocols and in vitro essential oil production have already been described in scientific literature. None of all scientific papers evaluated the effects of UHD substances on in vitro development or secondary metabolic production. The main goal was to evaluate the use of plant tissue culture to investigate the effects of UHD benzilaminopurine (BA) on Lippia alba shoot culture. Nodal segments obtained from plants growth in vitro was subcultured to Murashigue & Skoog semi-solid medium added with 2ml of these different solutions: BA 3µmol, BA 12CH (10-24), water 12CH and water (no dilution and succussion). Weekly 1 ml of solutions were added to cultures. The experiment was repeated twice and each one consisted in 3 culture vessel with 5 nodal segments per treatment (n=30). All plants were maintained in growth room under controlled temperature (25°C), light and photoperiod (16L/8D). The tested substances were prepared according to the method of stepwise dilution and succussion as describe in Brazilian Homeopathic Pharmacopoeia. The experiment was blinded all the time. After 60d, plantlets were evaluated for number of shoots, shoot length, rooted plants (%), callus development (%) and fresh biomass. Data were submitted to ANOVA following by Duncan’s and t-test. Plants from water 12CH and BA 12CH increased the number of new shoots and promoted the highest shoot length. By adding BA 3µmol the organogenetic response was inhibited since neither shoot nor root were developed. However, it was observed a significant basal callus development. Plant tissue culture could be adapted for UHD studies. More studies are being conducted in way to analyze other experimental conditions and biochemical/phytochemical parameters.

An Overview of Oil Palm Cultivation via Tissue Culture Technique

During the last three decades, plant cell, tissue, and organ culture have developed rapidly and become a major biotechnology tool in agriculture, horticulture, forestry, and industry. Many problems in conventional breeding techniques were solved via tissue culture techniques. Plant tissue culture technique permits the growing plants in test tube or closed container in vitro under controlled environment. This technique is devoted to solve two problems: 1) To keep the plant cells free from microbes. 2) To grow the desired plants by providing suitable nutrient medium and other environmental conditions. In this chapter, a review around plant tissue culture techniques that have been reported on oil palm breeding programme will be discussed. It is including the laboratory techniques, advantages and disadvantages of the technique, the problems to produce good and prolific oil palm tissue culture clones and mitigation measures that have been reported to overcome the problems. As a conclusion, this chapter reviews tissue culture techniques that could be used to propagate oil palm clones.

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In vitro plant tissue culture: means for production of biological active compounds

Claudia a. espinosa-leal.

Tecnologico de Monterrey, Campus Monterrey, Escuela de Ingeniería y Ciencias, Ave. Eugenio Garza Sada 2501, 64849 Monterrey, NL México

César A. Puente-Garza

Silverio garcía-lara, main conclusion.

Plant tissue culture as an important tool for the continuous production of active compounds including secondary metabolites and engineered molecules. Novel methods (gene editing, abiotic stress) can improve the technique.

Humans have a long history of reliance on plants for a supply of food, shelter and, most importantly, medicine. Current-day pharmaceuticals are typically based on plant-derived metabolites, with new products being discovered constantly. Nevertheless, the consistent and uniform supply of plant pharmaceuticals has often been compromised. One alternative for the production of important plant active compounds is in vitro plant tissue culture, as it assures independence from geographical conditions by eliminating the need to rely on wild plants. Plant transformation also allows the further use of plants for the production of engineered compounds, such as vaccines and multiple pharmaceuticals. This review summarizes the important bioactive compounds currently produced by plant tissue culture and the fundamental methods and plants employed for their production.

Introduction

Plant cell and tissue culture uses nutritive culture media and controlled aseptic conditions for the growth of plant cells, tissues and organs. Since its first establishment by Haberlandt in the early twentieth century, this type of culture has evolved into an essential tool for plant research at both the basic and applied levels (Haberlandt 1902 ). In vitro culture techniques are now indispensable for the production of disease-free plants, rapid multiplication of rare plant genotypes, plant genome transformation, and production of plant-derived metabolites of important commercial value (see Fig.  1 ) (Debnarh et al. 2006 ; Altpeter et al. 2016 ).

Diagram of current methods employed for the large-scale production of bioactive compounds using plant in vitro tissue culture

Due to the diversity of the methods and applications of available culture techniques, the subject of plant cell/tissue culture is extensively covered in the existing literature. Some works have even focused on the use of in vitro tissue culture for the production of secondary metabolites (Verpoorte et al. 2000 , 2002 ; Smetanska 2008 ; Karuppusamy 2009 ). This work aims to provide an updated overview on the use of in vitro culture for the production of medicinally or commercially important plant metabolites and bioengineered products, nevertheless because of the ample range of information available not all works within the scope of the article could be included, we apologize to those authors. The objective of this review is, therefore, to summarize the main molecules currently being produced using plant cell/tissue culture, their applications in areas such as medicine and food technology, and the plant material cultured for their production. The review also covers new trends in in vitro cell/tissue culture and plant transformation.

Production of biologically active compounds

Secondary metabolites.

The term secondary metabolite refers to a compound produced by plants, microorganisms or animals that is not required for their growth (Pickens et al. 2011 ). Humans have long used the products of plant secondary metabolism to satisfy a multitude of different needs (Borchardt 2002 ; Patwardhan 2005 ; Cragg and Newman 2013 ). The primary use of these compounds has been as medicinal agents, first in an empirical way and subsequently, starting in the 19th century, in a more rational way following the advent of molecule isolation (Corson and Crews 2007 ; Zenk and Juenger 2007 ; Cragg and Newman 2013 ). Despite the extensive research into secondary metabolites over such a long period of time, current estimates indicate that only about 6% of higher plants (between 300,000 and 500,000 species) have been systematically studied for their pharmacological potential, and only 15% have been evaluated for phytochemicals in general (Fabricant and Farnsworth 2001 ; Cragg and Newman 2013 ). Therefore, enormous opportunities exist for continued studies in this field.

The identification and isolation of a useful bioactive compound immediately generates a need for a method for its continuous production. A secondary metabolite is typically characterized by its diverse and complex chemical structure, usually encompassing multiple chiral centers and labile bonds, and this makes its chemical synthesis challenging (Pickens et al. 2011 ). Therefore, biologically active molecules are more commonly extracted from their natural sources. However, since most of the source plants are wild rather than domesticated species, harvesting from their natural habitats presents a risk of overexploitation, as well as creating a bottleneck in the production of the compounds. Further complications include the slow growth rates of many source plants, the low concentrations of the active compounds of interest and, frequently, the need for biotic or abiotic stress to induce biosynthesis. All these factors make the extraction of secondary metabolites from source species highly inefficient (Atanasov et al. 2015 ; Ochoa-Villarreal et al. 2016 ) and emphasize the need for novel approaches for secondary metabolite production.

The in vitro culture of plant cells and tissues under controlled conditions offers a well-founded technology platform for the production of plant natural products. The in vitro propagation (micropropagation) of plants or the in vitro culture of plant organs (usually roots) or callus can typically provide plant material capable of producing secondary metabolites (Atanasov et al. 2015 ; Morales-Rubio et al. 2016 ; Ochoa-Villarreal et al. 2016 ; Espinosa-Leal et al. 2017 ). Micropropagation has, therefore, become a commercially lucrative enterprise and provides marked advantages over conventional horticultural propagation practices by facilitating the production of large numbers of homogenous plants year-round, the generation of disease-free propagules and a substantial enhancement of multiplication rates (Debnarh et al. 2006 ). Currently, a large number of protocols are available for the micropropagation of medicinal plants (Debnarh et al. 2006 ; Rizvi and Kukreja 2010 ; Sarasan et al. 2011 ; Kaul et al. 2013 ; Kun-Hua et al. 2013 ; Bhattecheryya et al. 2014 ; Chen et al. 2014a ; Atanasov et al. 2015 ), as well as some commercially important plants, such as Agave salmiana (Puente-Garza et al. 2017a ), artichoke (Pandino et al. 2017 ), Stevia rebaudiana (Ramírez-Mosqueda et al. 2016 ) and Moringa oleifera (drumstick tree) (Juan-jie et al. 2017 ). However, the high costs of micropropagation compared with its traditional counterpart (i.e., collection from the wild) and the unpredictability of the needs of the market have limited the use of micropropagation at a commercial level (Debnarh et al. 2006 ; Methora et al. 2007 ; Lubbe and Verpoorte 2011 ; Pence 2011 ; Sahu and Sahu 2013 ). Notably, however, recent micropropagation efforts have been aimed at the conservation of overexploited medicinal plants, with special emphasis in plants used for traditional medicines in China and India (Rizvi and Kukreja 2010 ; Verma et al. 2012 ; Bhattecheryya et al. 2014 ; Chen et al. 2016a ).

Recent advances in plant cell culture, brought about by the established experience with microbial and animal cell culture, has resulted in effective scale-up from the experimental stage to an industrial scale. Plant cell culture now represents an efficient way to produce several valuable natural products (Fischer et al. 2015 ). As shown in Table  1 , the range of commercially important products includes pigments (e.g., anthocyanins and betacyanins), anti-inflammatory agents (e.g., berberine and rosmarinic acid), and anti-cancer molecules (e.g., paclitaxel and podophyllotoxin).

Table 1

List of some secondary metabolites industrially produced by plant tissue culture (with information of Wilson and Roberts 2012 ; Ochoa-Villarreal et al. 2016 )

Plant-made pharmaceuticals and other bioengineered products

In the early 1990s, transgenic plants were endorsed as an alternative means of production of pharmaceutically important proteins. A transgenic system offers several advantages, including decreased costs, increased ease of delivery and scale-up, decreased risk of contamination with animal and human pathogens, and eukaryotic protein processing. Despite the potential of the method, the past 20 years since its introduction have seen transgenic biomolecules (mostly orally delivered, plant-made vaccines) continue to languish in Phase I of human clinical trials. Only in this current decade has a breakthrough been made, and plant-based products are now geared to proceed to Phase II trials and beyond (Pogue et al. 2010 ; Thomas et al. 2011 ).

Table  2 lists the first plant-made human recombinant therapeutic protein approved by regulatory agencies for commercial sale in 2014. Developed by Pfizer, Inc. and Protalix Biotherapeutics, the enzyme taliglucerase alfa (commercially known as ELELYSO-™) is a treatment for Type 1 Gaucher disease (Pfizer 2014 ; Pastores et al. 2016 ). More plant-based products are awaiting approval in one of the phases of clinical trials, and some companies, including Icon Genetics, Ventria Bioscience and Greenovation Biotech, now have the technologies available for the production of several pharmaceuticals derived from plants (see Table  2 ). This seemingly show growth of the field can be attributed to some of the challenges presented by the method, such as low yields, unwanted glycosylation of products, purification and downstream processing hurdles, and the challenges inherent in the creation of a new manufacturing industry (Tusé et al. 2014 ; Yao et al. 2015 ).

Table 2

List of some pharmaceuticals produced in plants

With information of Wilson and Roberts ( 2012 ) and Yao et al. ( 2015 )

Some of these problems can be resolved by judicious selection of the plant material. For instance, the use of maize or lettuce, which are both edible and free of harmful substances, can reduce the need for the intensive purification required for the preparation of parenteral pharmaceuticals, thereby reducing the downstream costs (Hayden et al. 2012 , 2014 ; Lakshmi et al. 2013 ; Czyz et al. 2014 , 2016 ; Sue et al. 2015). In addition, the appropriate selection of the desired transgenic phenotypes and the use of breeding methods such as backcrossing or inbreeding can lead to the preservation of valuable traits, including high expression of the desired pharmaceutical by the plant material (Pniewski et al. 2017 ). Other advances have been made in glyco-engineering of host plants, which allows the engineered plants to produce human and mammalian-analogous molecules that exhibit comparable activity to their equivalents produced by mammalian cells in culture (Castilho et al. 2011 ; Zeitlin et al. 2011 ; Gleba et al. 2014 ).

Other plant metabolites of interest, which can be bioengineered, are plant growth regulators. These are typically produced in miniscule quantities in plants, but are essential for the regulation of plant cellular processes (Wani et al. 2016 ). Growth regulators also have critical roles in controlling plant responses to the abiotic stresses, such as drought, salinity and extreme temperatures, factors that limit crop productivity worldwide (Wani and Sah 2014 ). Due to these characteristics engineered plant growth regulators can be used for the improvement of crops from both nutritional and stress-resistance perspectives (Wani et al. 2016 ).

Plants employed for the production of biologically active compounds

An abundance of options, ranging from model plants and crops to wild-type plants, is available when selecting an appropriate plant material for the production of biomolecules. The selection of which plant to use will depend on a number of factors, with the chief considerations being the purpose of the study and the availability of the plant.

Most plants employed for the production of secondary metabolites are non-crop wild types, since these are the plants that already contain the metabolic tools for synthesis of many metabolites of interest (see Table  1 ). Some examples include Agave salmiana for saponins (Puente-Garza et al. 2017a ), Rhaponticoide mykalea for chlorogenic acids (Hayta et al. 2017 ) and phenolic compounds (Karalija et al. 2017 ), Leucophyllum frutescens for phenolic compounds (Espinosa-Leal et al. 2015 ) and Poliomintha glabrescens for luteolin (García-Pérez et al. 2012 ). All of these compounds are useful in the food, pharmaceutical or cosmetic industries (Perassolo et al. 2017 ). Other crop plants are used for the production of metabolites such as betalains, which are natural colorants as well as good antioxidants. The main sources of betalain metabolites are the cactus pear ( Opuntia ficus - indica ) and red beet ( Beta vulgaris ), and these metabolites are typically produced using hairy roots in bioreactors or by callus culture (Georgiev et al. 2008 ).

Other crop plants, or their parts, that are used for the production of bioengineered compounds, include carrot cells (enzyme replacement), rice (albumin serum) (both presented in Table  2 ), maize (vaccines, such as anti-HBV) and lettuce (vaccines and antibodies) (Hayden et al. 2012 , 2014 ; Lakshmi et al. 2013 ; Czyz et al. 2014 , 2016 ; Su et al. 2015 ; Yao et al. 2015 ). Model plants, such as Nicotiana benthamiana, are also transformed for the production of plant pharmaceuticals (Yao et al. 2015 ), as summarized in Table  2 .

Methods of plant tissue culture

Several methods are available for plant tissue culture; two of the most commonly used are presented in Fig.  1 . Organogenesis, which refers to the production of plant organs (roots or shoots), can be accomplished directly from meristems or indirectly from dedifferentiated cells (callus). The resulting cultures can later be used for the massive production of plants (micropropagation) or for the growth of particular organs (i.e., roots in hairy root culture). Callogenesis produces an amorphous mass of cells in response to exposure of explants to different growth regulators. The callus can then be used to regenerate whole plants, or it can be scaled up for the production of important metabolites in cell suspension cultures (Morales-Rubio et al. 2016 ).

All plant tissue culture methods follow a series of steps, as mentioned in Table  3 . First, the plant of interest needs to be selected; this is generally dependent on the aim of the study, but disease- and insect-free plants are preferred; if the plant requires it, some pre-treatments (fungicides and pesticides) can be applied. The next step is the initiation of the in vitro culture. The process requires the excision of small plant pieces (explants) or the use of seeds and their surface sterilization with chemicals. The explants are then placed in appropriate culture media and incubated for a short period of time; contaminated explants are discarded while the surviving ones continue to the next step. The following steps vary depending on the type of culture desired. In organogenesis, this is the propagation phase when explants are cultured on appropriate culture media for shoot or root multiplication, similarly in callogenesis the callus is multiplied. In the next step, callus and root cultures are scaled up for their culture using bioreactors, while the propagated shoots are transferred to root-promoting culture media, in the case of micropropagation. Finally, the micropropagated plants are hardened to grow individual plants capable of photosynthesis. The hardening is done gradually allowing the plants to acclimate to ex vitro conditions. Typically the plants are taken from high to low humidity and from low to high light intensity (Ahloowalia et al. 2003 ; Espinosa-Leal et al. 2017 ).

Table 3

List of in vitro tissue culture steps

With information from Ahloowalia et al. ( 2003 ) and Espinosa-Leal et al. ( 2017 )

The following sections cover novel developments in plant tissue culture that allow a more efficient production of bioactive compounds.

Improvements in traditional culturing techniques

Micropropagation has been the technique of choice for the production of whole plants for medicinal, conservation, reforestation and commercial purposes (Afolayan and Adebola 2004 ; Debnarh et al. 2006 ; Sarasan et al. 2011 ). Tissue culture of plants capable of producing important biomolecules offers a number of advantages over traditional field culture, including independence from geographical, seasonal, and environmental variations; uninterrupted production in uniform quality and yield; no need for pesticide and herbicide application; and comparatively short growth cycles (Rao and Ravishankar 2002 ; Debnarh et al. 2006 ). Production of secondary metabolites by plant tissue culture depends on a number of factors, chief among them are the nutrients provided for plant growth. The optimum nutrient concentration is a critical determinant in the growth of the explants and the accumulation of secondary metabolites (Rao and Ravishankar, 2002 ; Nagella and Murthy 2010 ; Murthy et al. 2014 ; Fargoso Monfort et al. 2018 ). The type of culture media used, the salt strength of the medium employed and the growth regulators, type and concentration used, are key factors that most be established for every culture (Rao and Ravishankar, 2002 ; Fargoso Monfort et al. 2018 ). The concentration of salts present in the culture media needed by a specific plant varies depending on the particular culture needs. The selection of a suitable medium is essential to establish cell and organ cultures (Nagella and Murthy 2010 ; Fargoso Monfort et al. 2018 ). Three of the most popular culture media employed are MS, B5 and WPM, as listed in Table  4 . MS basal media contains the highest total salts and nitrogen content. Nitrogen is an essential element that promotes explant growth, since it directly affects amino acid and nucleic acid production in the cells. MS basal media (1962) is the most commonly used plant culture media. As presented in Table  4 , it has a very high salt content, nevertheless, it is the preferred medium for the growth of several species (Alvarenga et al. 2015 ; Grzegorczyk-Karolak et al. 2015 ; Rahman et al. 2015 ). It has been demonstrated that different MS salt concentrations influence growth in several species (Assis et al. 2012 ; Martins et al. 2015 ; Shekhawat et al. 2015 ; Singh et al. 2015 ). Generally, lower salt concentrations stimulate rooting (Sorace et al. 2008 ; Golle et al. 2012 ; Shekhawat et al. 2015 ). Fargoso Monfort et al. ( 2018 ) found that in Ocimum basilicum volatile constituents diminished with higher salt concentrations. Plant growth regulators can affect the production of secondary metabolites, a recent review by Jamwal et al. ( 2018 ) presents a summary of the production of natural products by using different plant growth regulators.

Table 4

List of commonly used culture media used for in vitro plant tissue culture

With information from Fargoso Monfort et al. ( 2018 )

Nevertheless, one of the principal disadvantages of in vitro tissue culture is the high cost involved with the technique, particularly the expenses associated with culture media (mainly the carbon source, gelling agent and growth regulators), electricity and labor. Some studies have attempted to remedy the cost of culture media using alternative materials, such as household sugar or other sugars, as carbon sources and various types of starches and plant gums instead of agar. Other alternatives have included the use of liquid media and cell-suspension cultures, temporal immersion systems, and reusable glass beads as substitute support matrices (Etienne and Berthouly 2002 ; Goel et al. 2007 ; Thorpe 2007 ; Sahu and Sahu 2013 ). The cost of electricity can amount up to 60% of tissue culture production costs. The electrical energy is mostly employed for autoclaving, lighting of the growth room and air filtration in laminar-flow cabinets and air conditioning (Ahloowalia and Savangikar 2003 ; George and Manuel 2013 ). The use of artificial lighting in growth rooms is the most expensive and inefficient method on tissue culture technology. It generates heat that needs to be dissipated using air conditioning and it does not match natural light. Additionally, even though plants are capable of adapting to an ample range of conditions, once the adaptation occurs, re-adaptation to new conditions is slow and difficult (Ahloowalia and Savangikar 2003 ; George and Manuel 2013 ). The use of natural light is a low-cost option for tissue culture, it reduces electricity and capital costs as well as improves plant quality. There are several ways to achieve this, a simple option is to diffuse natural light under plastic or glass, this works best in temperate climates; some laboratories can be modified to adapt the use of ‘solatube’ which redirects daylight from rooftops through reflecting tubing (Kodym and Zapata-Arias 1999 , 2001 ; Kodym et al. 2001 ; Ahloowalia and Savangikar 2003 ) some laboratories can incorporate southwest facing windows in the growth rooms that allow for indirect diffused natural daylight, as is the case of bio-factories in Cuba (Baezas-Lopez 1995 ; Ahloowalia and Savangikar 2003 ) and finally some cultures have been successfully propagated using plastic bags as culture containers and hanging them in greenhouses thus eliminating the need of air-conditioned growth rooms (Ahloowalia and Savangikar 2003 ). Temperature regulation, another high electricity demanding feature of plant in vitro culture, can be mostly avoided since many plants can tolerate wide fluctuations in temperature (Kodym et al. 2001 ). Labor is another source of high in vitro tissue culture costs. Once the efficiency of labor in transferring number of propagules per hour has been achieved to the maximum possible, there is little room for improvement unless an automated or semi-automated system is implemented. For example, the use of bioreactors and mechanized handling of propagules, such systems have shown to reduce the cost of production by 50% (Ahloowalia and Savangikar 2003 ; George and Manuel 2013 ).

Another problem found in plant tissue culture involves the genetic stability of the plants. Different cases of micropropagation of in vitro-regenerated plants have shown that they are not always clonal copies of the mother plant (Devi et al. 2014 ; Bhattacharyya et al. 2017a ). In vitro culture conditions, especially some growth regulators and elicitors, act as stress factors that induce alterations in sensitive regions of plant genome, and therefore, generate instability in cultured cells, tissues and organs, an occurrence known as somaclonal variation (Larkin and Scowcroft 1981 ; Gyulai et al. 2003 ; Bairu et al. 2011 ; Stanišic et al. 2015 ; Govindaraju and Arulselvi 2016 ). The genetic changes experienced by the culture include: alternative DNA methylation, amplification, activation of transposable elements, polyploidy, changes in chromosome number or DNA sequence (Bairu et al. 2011 ; Stanišic et al. 2015 ; Govindaraju and Arulselvi 2016 ). Typically regeneration protocols involving a callus phase are considered the least reliable for clonal propagation, while plantlets regenerated by branching of the axillary buds or direct somatic embryos are considered to be, genetically, the most uniform (Rani and Raina 2000 ; Varshney et al. 2001 ; Bhattacharyya et al. 2017b ). The occurrence of somaclonal variations during in vitro propagation, industrial production of phytochemicals, or genetically engineered plants can lead to massive economic consequences and represents a serious obstacle in the practical utilization of plant tissue culture techniques for the production of active metabolites (Rahman and Rajora 2001 ; Bhattacharyya et al. 2016 ). Therefore, to screen somaclonal variability within a cell culture, it is necessary to monitor and assess the genetic constitution and stability of the in vitro-regenerated plants. The methodologies involved in that process include the use of several techniques to assess possible alterations at different levels (Devarumath et al. 2002 ; Bhattacharyya et al. 2015 , 2017a ; Bose et al. 2016 ; Bhattacharyya and Van Staden 2016 ). Flow cytometry and chromosome counting are widely used to assess changes in ploidy and chromosome number; PCR-based DNA markers, random amplified polymorphic DNA (RAPD), random fragmented length polymorphism (RFLP), inter simple sequence repeat (ISSR), amplified fragment length polymorphism (AFLP), microsatellite markers, and start codon targeted (ScoT) polymorphism have been successfully used to evaluate genomic stability of regenerated plants (Hu et al. 2008 ; Collard and Mackill 2009 ; Bairu et al. 2011 ; Singh et al. 2013 ; Bhattacharyya et al. 2014 , 2015 ; Rathore et al. 2014 ; Stanišic et al. 2015 ; Bose et al. 2016 ; Govindaraju and Arulselvi 2016 ). An appropriate combination of two or more markers guarantees reliable and efficient testing of genetic fidelity in plants (Palombi and Damiano 2002 ; Bhattacharyya et al. 2016 ).

Cell suspension culture remains the best method for the production of active metabolites, especially natural products, with paclitaxel from Taxus spp. being the most prominent example (Atanasov et al. 2015 ) (Table  1 ). For the production of cell suspension cultures, the calli are first induced in solid media, the cells are then transferred to liquid media. In small laboratories, the cells are first grown in shaking flasks and later transferred to large-scale liquid-phase bioreactors. There are many different types of reactors including tank reactors, bubble beds and rotary rectors (Furusaki and Takeda 2017 ). Comprehensive reviews on industrial bioreactors have been published previously and can be consulted for more information on the subject (Su and Lee 2007 ; Huang and McDonald 2012 ).

A number of limitations are associated with these methods, when compared with microbial cultures, such as the slow growth rates and low and variable yields of metabolites, and these limitations still restrict the industrial use of plant cell suspension cultures (Kolewe et al. 2008 ; Kirakosyan et al. 2009 ). However, the production of important metabolites can be enhanced with modifications to the culture media, such as the addition of elicitors or precursors, or the environmental conditions.

Elicitors stimulate the production of plant natural products that serve as plant defense compounds. Several types of elicitors can increase the secondary metabolite production, including pectin and cellulose (plant cell wall constituents), chitin and glucan (from microorganisms) and salicylic acid and methyl jasmonate (plant immune signaling molecules) (Namdeo 2007 ; Shilpa et al. 2010 ; Sharma et al. 2011 ; Ochoa-Villarreal et al. 2016 ). Srivastava and Srivastava ( 2014 ) used different fungal culture filtrates as biotic elicitors in root cultures of Azadirachta indica to promote the production of Azadirachtin. Filtrates of Curvularia lunata yielded the highest production of the target compound compared to control. Saranya Krishnan and Siril ( 2018 ) used yeast extracts, pectin and xylan to elicit the production of anthraquinones in Oldenlandia umbellate cultures, with the addition of pectin resulting in the highest elicitation. The selection of the adequate elicitor will depend on the metabolite being produced and the plant culture employed.

Another strategy is to supplement with precursors that are intermediary compounds of the metabolic pathway of the desired natural product. Supplementing the culture media with precursors of secondary metabolites can enhance the yield of the final product (Rao and Ravishankar 2002 ; Hussain et al. 2012 ), and has been used successfully in several cases, including the production of phenolic compounds (Palacio et al. 2011 ), triterpenoids (Chen et al. 2016b ) and withanolides (Sivanandhan et al. 2014 ). Cultures of Antrodia cinnamomea were fed exogenous sterols including squalene, cholesterol, and stigmasterol to enhance their triterpenoid content, the feeding of high doses of stigmasterol resulted in an increased amount of terpens (Chen et al. 2016b ). When searching for appropriate precursors it is important to look at the entire biosynthetic pathway and include several molecules involved in different steps of the process, incorporating some examples that affect the production of the target compound in indirect ways. Srivastava and Srivastava ( 2014 ) studied the use of precursors to enhance production of azadirachtin including sodium acetate, cholesterol, squalene, isopentenyl pyrophosphate, and others. They found the best results using cholesterol as an indirect precursor. Parra et al. ( 2017 ), evaluated the effect of different biochemical precursors in fatty acids production in cacao cell cultures including biotin, pyruvate, acetate and bicarbonate, they also utilized glycerol because it is involved in triglycerides assembly. The later was found to induce a higher fat production compared to the other precursors and control. It is also important to remember that the precursor must be easier and cheaper to acquire the compound of interest.

An alternative way of increasing natural product production is by manipulation of environmental factors. Plants are heavily influenced by environmental factors to regulate the biosynthesis of secondary metabolites, through stress response mechanisms (Zhi-lin et al. 2007 ; Verma and Shukla 2015 ). Abiotic stresses related to environmental factors include light intensity, water availability, temperature (high or low), radiation (UV), gaseous toxins (ozone), pesticides and metals (Ni, Cd, Co, Fe, Zn) (Ramakrishna and Ravishankar 2011 ; Raduisene et al. 2012 ). Water stress is produced when the plant has limited water availability, either because there is no water (drought) or because the water is dissolving a solute (salinity). Drought stress can be induced in vitro by the addition of high-molecular-weight solutes, such as polyethylene glycol (PEG), to the culture media (Verslues et al. 2006 ). It has been proven to enhance the production of saponins in A. salmiana (Puente-Garza et al. 2017b ), phenolics in Poliomintha glabrescens (García-Pérez et al. 2012 ) and rosmarinic acid in Salvia miltiorrhiza (Liu et al. 2011 ). For the induction of salinity stress different concentrations of salts, such as NaCl and Na 2 CO 3 , can be added to the culture media. This type of stress has resulted in increased production of steviol glycoside content in Stevia rebaudiana (Gupta et al. 2016 ); Sorbito in Lycopersicon esculentum (Tari et al. 2010 ) and flavonoids in Hordeum vulgare (Ali and Abbas 2003 ). Light is an essential abiotic environmental component for plants, since it permits photosynthesis; however, high levels of UV radiation can be harmful (Verma and Shukla 2015 ). To protect itself form oxidative damage the plant produces several antioxidant metabolites such as polyphenols and tocopherols (Kaur and Kapoor 2001 ; Argolo et al. 2004 ). UV radiation can, therefore, be used to enhance the production of secondary metabolites, typically by exposing the cultures to UV light using a special lamp for a set period of time. The technique has been used in Vitis vinifera calli to produce flavonols (Cetin 2014 ). Different chemicals, including heavy metals, can cause chemical stress. Metal ions influence the production of secondary metabolites, and depending on the plant species and heavy metal type and concentration can even enhance their production (Ramakrishna and Ravishankar 2011 ; Verma and Shukla 2015 ; Maleki et al. 2017 ). As an example is the case of Thalictrum rugosum cell suspension culture that used CuSO 4 to stimulate the production of berberine (Kim et al. 1991 ).

These improvements, however, are not sufficient to sustain an adequate large-scale production of bioactive compounds, so other methods are still needed.

Alternatives to in vitro plant culture

The low yield of secondary metabolites in cell cultures can be explained as a consequence of a lack of cell differentiation. An alternative strategy to cell culture is the organized culture of roots or shoots (Verporte et al. 2002; Kolewe et al. 2008 ). Hairy root culture is a perfect example; it is induced by infection of roots with Agrobacterium rhizogenes and the subsequent transfer of the Ri plasmid, which induces abundant growth of neoplastic roots that can be maintained in vitro (Ron et al. 2014 ). Grzegorczyk-Karolak et al. ( 2018 ) obtained hairy root culture of Salvia viridis by wounding shoot explants with needles dipped in Agrobacterium rhizogenes strain A4 culture. Some of the advantages provided by hairy root cultures include high growth rates without the need for plant growth regulators, genetically and biochemically stable cultures, and a similar capacity for production of secondary metabolites than cell suspension cultures (Guillon et al. 2006 ; Georgiev et al. 2012 ; Mora-Pale et al. 2014 ). A wide range of natural products has been produced using this system, including lignans, steroids, anthraquinones and alkaloids (Doma et al. 2012 ; Huang et al. 2014 ; Pandey et al. 2014 ; Wawrosch et al. 2014 ). However, metabolite production requires that the compound of interest be one that is normally synthesized within the roots of the source plant, so this limits the versatility of the hairy root system (Ochoa-Villarreal et al. 2016 ).

Nevertheless, the greatest limiting factor in the industrial use of hairy rots for the production of secondary metabolites is their scale-up, since it requires the development of appropriate culture vessels that permit mixing without causing shear damage to the interconnected root mat. Binoy et al. ( 2016 ) designed a novel culture vessel by customizing a reaction kettle (2 L) for the culture of Plumbago rosea and production of plumbagin an anticancer molecule. Srivastava and Srivastava ( 2007 ) described the different configurations and parameters of bioreactors for hairy roots culture, which include liquid phase, gas phase and hybrid systems. ROOT Bioactives AG, a Swiss company, has successfully developed and optimized a bioreactor system for large-scale cultivation of hairy roots capable of producing many kinds of bioactive compounds, including secondary metabolites and recombinant glycosylated proteins with pharmaceutical properties (Atanasov et al. 2015 ; Ochoa-Villarreal et al. 2016 ). However, many factors influence the scale-up process, such as culture medium properties, hormonal balance, gaseous composition, growth kinetics, inoculum density, culture period, and the species (Ho et al. 2017 ). In the scale-up process, Ho et al. ( 2017 ) recently obtained a 4.01-kg dry root biomass with a yield of 287.12 mg/L of total phenolic productivity for Polygonum multiflorum in a 500 L pilot-scale reactor. Hybrid systems, which include a liquid- and a gas phase, are reactors that attend the disadvantage of the null distribution uniformity of cells in gas-phase reactors, and the limitations of the mass transfer in liquid-phase reactors; these systems offer the best compromise between the two systems, used in alternate way (Srivastava and Srivastava 2007 ). Biomass measurement is one of the major challenges during scale-up of hairy roots culture, however, it is also important to determine the harvest capacity of secondary metabolites and their bioactivity, some examples are phenolic compounds (Ho et al. 2017 ; Thiruvengadam et al. 2014 ), xanthones (Vinterhalter et al. 2015 ), glucosinolates (Chung et al. 2016 ), tetraterpenoids (Thakore and Srivastava 2017 ), among others.

Transformed hairy roots have shown a higher biological activity, compared with non-transformed roots (Chung et al. 2016 ; Jeong et al. 2005 ; Vinterhalter et al. 2015 ). However, it is important to determine the optimal number of subcultures to avoid somaclonal variations that can affect the yield and behavior of plant cell cultures (Martínez-Estrada et al. 2017 ). Additionally, hairy root culture is susceptible to the use of biotic and abiotic elicitors to enhance the production of the desired metabolites, like the examples presented above from Srivastava and Strivastava (Srivastava and Srivastava 2014 ). Furthermore, a combined approach using both types of elicitors can be employed. For instance Wang et al. ( 2016 ) used ultraviolet-B (UV-B) radiation and methyl jasmonate applied alone or in combination in Salvia miltiorrhiza hairy root cultures and found that the combined treatment exhibited synergistic effects on the expression levels of genes in the tanshinone biosynthetic pathway.

Another way to avoid some of the major difficulties encountered when using cell cultures, such as variability in product biosynthesis, large cell aggregates and shear stress (Yun et al. 2012 ), is to use cultures of undifferentiated cambial meristematic cells. These cultures are not only free of the problems stated above, but the cells are physiologically stable and show high growth rates (Lee et al. 2010 ; Jang et al. 2012 ). Cambial meristematic cells are undifferentiated cells that grow indefinitely, behaving like plant stem cells (Lee et al. 2010 ; Ochoa-Villarreal et al. 2016 ). They eliminate the need for dedifferentiating plant cells for the establishment of plant cell cultures, and they offer greater stability in product accumulation over long periods (Lee et al. 2010 ). This type of culture has been established for the production of nutritional, cosmetic and medicinal products and is currently considered a key platform for the large-scale production of natural products (Roberts and Kolewe 2010 ; Yun et al. 2012 ; Ochoa-Villarreal et al. 2016 ).

Plant cell cultures rarely present uniform physiological characteristics, most especially callus cultures. Their heterogeneous nature results in the need to select highly productive cell lines to establish profitable production platforms for natural products (Mulabagal and Tsay 2004 ; Ochoa-Villarreal et al. 2016 ). A novel cell line can be obtained by employing cell-cloning methods. First, since accumulation of active metabolites is genotype specific, an appropriate selection of suitable species and later organs for callus production is needed (Murthy et al. 2014 ; Ochoa-Villarreal et al. 2016 ). The selection process depends on the type of compound intended to produce, if the product of interest is a pigment a spectrophotometric method can be employed, however, if it is not other chemical-based approaches are needed (Fujita et al. 1984 ; Mulabagal and Tsay 2004 ; Ochoa-Villarreal et al. 2016 ). A popular method involves the identification of cell lines that exhibit a high level of metabolic flux through the targeted pathway by the exogenous application of an intermediate (Ochoa-Villarreal et al. 2016 ). For example, high producing shoots of Mentha arvensis were screened by adding menthol to the culture media. The surviving clones exhibited high menthol tolerance, thus presenting genotypes capable of elevated menthol production (Dhawan et al. 2003 ). Nevertheless, the prolonged use of selected natural product producing cell lines is limited, since they often loose their ability to produce the desired metabolites (Georgiev et al. 2009 ; Wilson and Roberts 2012 ; Ochoa-Villarreal et al. 2016 ). The cell lines decrease or loss of active compound biosynthesis is due, in most cases, to genetic instability resulting from somaclonal variations (Ochoa-Villarreal et al. 2016 ).

Despite the problems presented above, there are some commercially available plant cell lines currently in the market. The most popular is Tobacco Bright Yellow-2 cells (BY-2), they are attractive because of their fast growth rate and their ease of Agrobacterium -mediated transformation and cell cycle synchronization (Su and Lee 2007 ). Additionally, the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures have an ample catalog of plant cell lines. They offer 41-plant cell lines, 18 of which are delivered as actively growing cultures and 23 are maintained as cryopreserved cultures. Among the plants offered are some model plants like Solanum tuberosum and Arabidopsis thaliana and some medicinal plants like Echinacea angustifolia, Arnica montana and Valeriana officinalis (Leibniz Gemeinschaft 2018 ). Acquisition of the cell lines would facilitate the establishment of the culture and allow for a more streamline process of plant transformation and metabolite production.

Strategies for the expression of plant biologically active compounds

Low expression levels of plant active metabolites and expression of new important compounds not typically expressed in plants, such as vaccines, create a need for tools that allow the modification of plant genetic material. Plant genetic engineering has been practiced since the 1980s, first with Agrobacterium and later with transformation mediated by particle bombardment. Both techniques have been effective for an array of plants, allowing over-expression of secondary metabolites or the production of plant-made pharmaceuticals. Nevertheless, despite this success, these methods present several challenges that limit their use in many crops (Altpeter et al. 2016 ).

Agrobacterium -mediated transformation can be performed in most dicotyledonous (dicot) plants (however, it is mostly limited to some genotypes within a species) and in a small number of monocotyledonous (monocot) plants (Klee et al. 1987 ; Nam et al. 1997 ). Problems arising from the use of A. tumefaciens for plant transformation include difficulties obtaining licensing (Chi-Ham et al. 2012 ), high costs of securing regulatory approval, and plant innate responses to bacterial infection, such as the activation of some proteins that cause tissue browning and necrosis that, in turn, reduce transformation frequencies (Altpeter et al. 2016 ). Some of these difficulties can be easily avoided with small modifications to the technique, like downregulating infection-responsive genes in the host plants or adding antioxidants to the infection medium (Altpeter et al. 2016 ).

To eliminate the rest of the problems, researchers continue to seek out a novel gene delivery system based on a non-pathogenic organism that will provide high rates of transformation for both dicot and monocot species. Advances have been made with several species of Rhizobium ( Sinorhizobium meliloti, Mesorhizobium loti and NGR 234) known as Transbacter (Zuniga-Soto et al. 2015 ). Another gram-negative bacterial member of the Rhizobiaceae family, Ensifer adhaerens strain OV14, unlike A. tumefaciens, also seems to be beneficial to plants and has been used successfully for the transformation of Arabidopsis thaliana, Solanum tuberosum and Oryza sativa L. (Martin 2002 ; Wendt et al. 2012 ; Zhou et al. 2013 ).

Bioballistics can be used on a wider range of plant genotypes than are amenable to Agrobacterium transformation. This technique lacks the pathogenic characteristic of the bacteria and simplifies the cloning process since it does not require a specific vector (Chen et al. 2014b ; Altpeter et al. 2016 ). However, plant tissues subjected to this technique often show difficulties in regeneration after bombardment and in the transgene performance. The effectiveness seems to depend on particle characteristics, such as type, size, quantity and acceleration; on the DNA amount and structure; and on the tissue type and pretreatment (Zuniga-Soto et al. 2015 ). Advancements in these techniques could potentially enhance the regeneration and transformation responses of a wide range of plants of economic interest (Zuniga-Soto et al. 2015 ).

Genome editing mediated by CRISPR/Cas9, a new development in plant genome transformation, may be a promising solution to most of the problems of the currently available techniques. This new technology allows modification of specific areas of the genome with an increased precision of the insertion, while preventing cell toxicity and offering perfect reproducibility (Voytas 2013 ; Voytas and Gao 2014 ). Genome editing is currently applied in one of three forms: (1) Alteration of a small number of nucleotides, (2) replacement of an allele with a pre-existing one, and (3) insertion of new genes in predetermined regions of the genome (Abdallah et al. 2015 ). Since genome editing techniques create only small traces of DNA alterations, most regulatory procedures associated with transgenic plants are avoided and the technique can be employed for the rapid creation of new crops with pest resistance, enhanced nutritional value and drought tolerance (Voytas 2013 ; Abdallah et al. 2015 ; Li et al. 2015 ). Some successful examples include transformations of solanaceous crop plants like potato and tomato (Van Eck 2018 ), soybean (Li et al. 2015 ) and some cereals, such as barley, maize, rice, wheat and sorghum (Zhu et al. 2017 ).

Perspectives for plant production of bioactive molecules

In vitro tissue culture is a vital tool that can be employed for the rapid production of important metabolites. Once a commercially important compound has been identified and isolated, measures can be taken for its scale-up production, as shown in Fig.  1 . Currently, several systems, such as suspension cultures and hairy roots, allow for the large-scale manufacture of plant compounds (Fig.  1 ) (Xu et al. 2012 ). Nevertheless, as mentioned earlier, the high costs associated with this technology makes it uncompetitive when compared to less expensive but environmentally unsustainable processes such as collection of wild plants or when compared to chemical synthesis. These culture methods also still require the use of sterilizable bioreactors, so their scale-up is limited (Nogueira et al. 2018 ; Buyel et al. 2017 ).

The advent of novel molecular tools now presents new possibilities for the production of important metabolites using plant systems. Chief among these is the use of targeted genome engineering, particularly the previously mentioned genome editing mediated by CRISPR/Cas9. The use of this technological approach creates the possibility of producing new plant varieties without the introduction of foreign genes (Doudna and Charpentier 2014 ; Baltes and Voytas 2015 ; Nogueira et al. 2018 ). Gene editing could potentially be used for the introduction of new alleles, promoter replacement or the introduction of new pathways, all of which could result in the creation of plant-based systems capable of novel expression of useful bioactive molecules (Nogueira et al. 2018 ).

Once the plant has been engineered (either by gene editing by CRISPR/Cas9 or by traditional methods), and has passed through in vitro culture, the genetic material can be stored using master and working seed banks (Sack et al. 2015 ). The plants can then be used for the large-scale production of the desired metabolites, including plant-made pharmaceuticals. Three options are available for the cultivation of the engineered plants: under open-field conditions, if legal, in conventional greenhouses or in vertical farming units, as modeled in Fig.  1 , with the latter two offering ease of scale-up and appropriate containment of the plants (Buyel et al. 2017 ).

Some plants, like medicinal plants, benefit from in vitro conditions (faster growth rates) and should, therefore, be sustained in those settings (Rao and Ravishankar 2002 ). With this in mind, several actions can increase the production of secondary metabolites by plant cells (Fig.  1 ); some of these, like the use of precursors and elicitors, have already been mentioned. The manipulation of environmental factors, including high/low temperature, drought, UV, alkalinity, salinity, exposure to heavy metals, and others, is now emphasized. These conditions, which are potentially damaging to the plants, often increase the capacity for production or even induce de novo synthesis of secondary metabolites in plant in vitro cultures (Korkina et al. 2017 ; Lajayer et al. 2017 ; Moon et al. 2017 ; Puente-Garza et al. 2017b ).

Conclusions

The use of in vitro tissue culture remains a feasible strategy for the production of structurally complex and high-value natural products, especially if the plant source material is an overexploited, slow-growing or low-yielding plant. However, due to the higher costs, a cost–benefit analysis of in vitro culture is wise before implementation of the technique. Similarly, the production of pharmaceuticals using plant culture systems can offer significant advantages, including reduction in costs, rapid production, low burden of human pathogens and scalability; all these advantages are plant product specific and depend on the production efficiencies compared to those offered by alternative sources. In the next decade, tissue culture should reach its full potential with the use of novel technologies such as gene editing and environmental factor manipulation.

Author contribution statement

All authors contributed equally to this work in terms of writing and conception. All authors wrote and reviewed the latest version of this manuscript.

Acknowledgements

The Research Nutriomics Chair Funds and CAT-005 from Tecnologico de Monterrey, Escuela de Ingeniería y Ciencias, as well as Postdoctoral fellowships presented to Dr. Claudia Espinosa-Leal by Consejo Nacional de Ciencia y Tecnología (CONACyT), Mexico and Tecnologico de Monterrey supported this research.

Compliance with ethical standards

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Nanomaterials in plant tissue culture: the disclosed and undisclosed.

* Corresponding authors

a Department of Bioresources and Food Science, Konkuk University, 1, Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea E-mail: [email protected] Fax: +82 24503310 Tel: +82 24500574

Plant tissue cultures are the core of plant biology, which is important for conservation, mass propagation, genetic manipulation, bioactive compound production and plant improvement. In recent years, the application of nanoparticles (NPs) has successfully led to the elimination of microbial contaminants from explants and demonstrated the positive role of NPs in callus induction, organogenesis, somatic embryogenesis, somaclonal variation, genetic transformation and secondary metabolite production. This review aims to consolidate all of the current achievements made through the integration of nanotechnology into plant tissue culture and highlight the positive attributes of using NPs in plant tissue culture. Both the positive and adverse effects of using NPs in the culture medium are discussed and presented. The toxicity aspects and the safety concerns of exposing plants and the associated environment to NPs are recorded. Finally, future prospects through the involvement of not merely Ag, TiO 2 , and ZnO NPs, but more recent innovations such as graphene, carbon nanotubes, SiO 2 , quantum dots, and dendrimers are proposed. The undisclosed shadows hanging in the background, including the repercussions of using nanomaterials without proper awareness, as well as dosage-based adverse effects and nanotoxicity aspects, are highlighted. The need for more research in the pursuit of discrete answers to unresolved questions regarding mechanisms is emphasized as the key to real progress in plant nanobiotechnology.

• This article is part of the themed collections: 2018 Open Access Week Collection , 2017 and 2018 RSC Advances Reviews from Around the World , 2017 Review articles and RSC Advances: Most downloaded articles of 2017

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Application of nanoparticles in plant tissue cultures: minuscule size but huge effects

• Published: 16 November 2023
• Volume 155 , pages 323–326, ( 2023 )

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• S. Ochatt 1 ,
• M. R. Abdollahi 2 ,
• M. Akin 3 ,
• J. J. Bello Bello 4 ,
• K. Eimert 5 ,
• M. Faisal 6 &
• D. T. Nhut 7  

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Reliable and efficient strategies for plant regeneration are the prerequisites for reproducible and successful propagation, conservation, gene transfer and enhanced secondary metabolite production in vitro. In this respect, treatments with nanoparticles (NPs) studied in recent years have successfully eliminated microbial contaminants from explants, with a parallel positive impact on callus proliferation, but also on the induction of organogenesis, somatic embryogenesis, somaclonal variation, in vitro conservation, genetic transformation, and secondary metabolite production.

This Special Issue (SI) of PCTOC focuses on this emerging in vitro technology, as well as on the study of the potential hormetic response, toxicity concerns and safety issues resulting from the use of NPs in plant tissue cultures. It includes three comprehensive review papers and sixteen original articles.

In an authoritative review, Inam et al. surveyed the literature concerning the use of metal oxide NPs as nano-elicitors for secondary metabolite production. Recent years have seen an increasing interest in the production and uses of metal oxide nanoparticles for various purposes, among which are the improvement of the production of secondary metabolites by cultured cells and callus of a range of species. Secondary metabolites accumulate in tissues as a defense reaction viz. a viz. of several abiotic stress agents, including salinity, drought, and extreme temperatures among others. In this review, the authors examined the different routes of exposure of metal oxide NPs in plants, and also their role as novel elicitors of important phenols, flavonoids, alkaloids, and terpenes, with relevant metabolic functions. Interestingly, they critically discussed the mechanism underlying nano-elicitation and NP uptake and translocation in plants, proposing future research directions.

A comprehensive review by Sena et al. discussed the applications of green synthesized NPs in medicinal plant research. This eco-friendly approach to produce NPs is a viable, quick, and effective strategy. The use of NPs has sometimes been suggested to encompass a certain level of toxicity due to the methods used for their obtention and green synthesis appears as the logical alternative to contour this but, rather surprisingly, though, it has only seldom been researched. The authors also delved into the significance and uses of NPs within the context of secondary metabolites production, as well as their notable antioxidant, antibacterial, and antimicrobial activities, which can also accelerate plant development, enhance photosynthetic efficiency, and improve the plant performance in general. They highlighted a possible hormetic effect or hormesis of the studied NPs on plant development, that can be defined as “a stimulatory process of low dose and inhibition at high doses of NPs”. It has been stated that low concentrations of NPs induce hormetic effects through activating plant stress defence mechanisms. This paper discusses how NPs act depending on the precise particle size, composition, concentration, and application method, areas that still require more research input for a better comprehension of the mechanisms underlying their action.

Humbal and Pathak summarized the state-of-the-art of the application of various metallic, bimetallic, non-metallic, carbon-based, and composite NPs as elicitors of economically important medicinal secondary metabolites in different species. They briefly explained the exposure, uptake, and translocation of nanoparticles inside the plant cell and discussed the possible mechanisms of nanoparticle-mediated elicitation of secondary metabolites in plant tissue cultures.

Of the various NPs used in plant tissue cultures, metal NPs have been more frequently studied, and among them silver NPs (AgNPs) are the most common in the literature. It is hence not surprising that five articles in this SI concerned the use of AgNPs with different species and for different purposes.

Truong et al. reported the enhancement of plant regeneration competence from leaf and internode explants through thin cell layer culture in purple passion fruit ( Passiflora edulis Sims f. edulis ) using AgNPs. They found that 1.5 mg L − 1 AgNPs associated with BAP favoured the largest regeneration responses from the leaf explants, while for internode explants there was a notable topophysis effect, whereby the position of the internode within the stem affected the regeneration competence of the explants thereof, likely correlated with the endogenous hormone concentration at different node positions. Moreover, it was found that the addition of 3.0 mg L − 1 AgNPs significantly enhanced the proliferation and maturation of somatic embryos from thin cell layer internode explants.

In another study from the same team, Cuong et al. showed that AgNPs significantly improved the micropropagation of Limonium sinuatum (L.) Mill. ‘White’, both for explant surface disinfection, but also for the in vitro growth, development and acclimatization of produced plants. Noteworthy, a modulating effect of AgNPs was recorded on endogenous hormone content during the shoot multiplication and rooting stages of plantlets. Explants treated with 200 mg L − 1 AgNPs for 20 min exhibited a better disinfection and shoot induction than those disinfected with 1000 mg L − 1 HgCl 2 for 5 min (of relevance in the wake of potential limitations for the use of HgCl 2 in several countries). The explants cultured in presence of 1.0 mg L − 1 AgNPs produced more shoots/explant than the control by reducing ethylene content and increasing zeatin content. Likewise, a medium with 0.4 mg L − 1 AgNPs shortened the delay for rooting of plantlets indicating a low ethylene content and high content of IAA, GA 3 , and ABA compared to the untreated controls, and the resulting plants also showed better greenhouse acclimatization than those of the control.

On the other hand, Manokari et al. reported that AgNPs improved the in vitro propagation responses of Gaillardia pulchella cv. ‘Torch Yellow’, with optimum shoot proliferation and shoot biomass for explants cultured on MS medium supplemented with 0.5 mg L − 1 BAP, 0.25 mg L − 1 IAA and 4.0 mg L − 1 biogenic AgNPs as compared with the control. In addition, shoots developed on AgNPs-containing medium were healthy, sturdy, and greener than the controls produced on media lacking AgNPs, that were hyperhydric and chlorotic. AgNPs enhanced chlorophylls and carbohydrate contents and reduced carotenoids in the leaves, also improving root induction and the acclimatization of in vitro propagated plantlets. This likely occurred through an improved organization of stomatal complexes and trichomes development which, in turn, enhanced the defence mechanism towards abiotic stress thereby helping the plantlets to survive during acclimatization and post-acclimatization under greenhouse and, later, field conditions.

Andújar et al. showed that AgNPs promoted dipertene production in Stevia rebaudiana cultures in temporary immersion bioreactors for 21 days. They showed that 25 and 37.5 mg L − 1 AgNPs decreased shoot multiplication rate, shoot length, the number of nodes and leaves per shoot, and their fresh and dry weights compared with the control, while no negative effect was observed at a lower (12.5 mg L − 1 ) concentration. On the other hand, chlorophyll a, carotenoids and soluble phenolics were increased in plants supplied with 25 mg L − 1 AgNPs, suggesting oxidative stress. The endogenous levels of diterpenes were significantly increased by the application of 12.5 mg L − 1 AgNPs. Altogether, the results indicate the potential role of AgNPs as elicitors to promote diterpenes production in stevia, provided a balance is ensured between oxidative damage and secondary metabolite production.

Working with five different in vitro grown crops, Tomaszewska-Sowa et al. examined the cyto- and genotoxic side effects of using AgNPs as antimicrobial agents instead of standard sterilization methods. They tested the effects of a range of 50 to 100 mg L − 1 AgNPs on endoreduplication, DNA content and growth of seedlings grown in vitro of rapeseed, white mustard, sugar beet, red clover, and alfalfa. The genome size and DNA synthesis patterns in the roots, hypocotyls, and leaves from seedlings of these species were established by flow cytometry. It was found that while AgNP-treatment did not influence germination or genome size, it did increase root length and endoreduplication intensity, which could be interpreted as a response defence mechanism against stress provoked by the disruption of mitotic division by AgNPs.

Yoshihara et al. studied the effects of overexposure to metal oxide NPs on the root elongation and chlorophyll production in lettuce. In this appealing work, the authors exposed lettuce seedlings to Zn applied as ZnNPs and Zn 2+ ion in aqueous solutions. Thus, 0.74 mg L − 1 Zn 2+ ions provided as 10 mg L − 1 ZnNPs inhibited root elongation while no such inhibition occurred when the same amount of Zn 2+ ions dissociated from ZnCl 2 was provided. Dispersions of water insoluble SiO 2 and TiO 2 NPs did not affect root elongation, which suggests that the phytotoxicity effect observed was due to the ionizable metal oxide ZnONP dispersions. Indeed, the Zn content in lettuce roots incubated in ZnONP dispersions was much higher than for ZnCl 2 solution-incubated roots. A 20 mg L − 1 ZnONPs dispersion reduced the chlorophyll contents of seedlings, and all plants died after transplanting onto a ZnONPs-free medium. Inhibition of root elongation was accompanied by an accumulation of water-soluble components of the cell walls in roots through a specific mechanism.

Also working with ZnONPs, Canales-Mendoza et al. evaluated the in vitro multiplication responses of Agave salmiana var. Ayoteco. They used ZnONPs of an average size of 70 nm biosynthesized from cell-free filtrate from Mucor fragilis . A 20-day treatment with ZnONPs promoted organogenesis and modified the structures in shoots and seedlings, mainly the stomata. This occurred without the accumulation of ZnO and was coupled with antioxidant activity in such tissues that depended on the stress generated by abiotic agents and on the NPs to which they were exposed.

In two independent articles, Hanif et al. reported the use of NPs to improve the drought tolerance induced by 5% and 10% PEG stress in Coriandrum sativum . In the first study, proline coated ZnONPs were assessed as a nanofertilizer against drought stress. The authors characterized by scanning electron microscopy (SEM) and powder X-ray diffraction (XRD) ZnONPs with hexagonal structures of 14.73 and 20.59 nm, which significantly increased the shoot and root length as well as the dry weight of plants grown under stress. Moreover, the biochemical and antioxidant profile of such plants demonstrated the stress alleviating effect of the ZnOPNPs, through a decreased contents of phenolics and flavonoids as NPs concentration increased. At 100 mg L − 1 , ZnOPNPs reduced the free radical scavenging activity in shoots and in root, while the total antioxidant profile decreased due to the improvement in antioxidant enzyme activity which reduced drought stress in the coriander plants. In a follow-up article, Hanif et al. studied the synergistic effect of a glycine betain-ZnO nanocomposite in coriander. Thus, ZnONPs were coated with glycine betaine (ZnOBtNPs), SEM and XRD showed that the ZnONPs were slightly smaller and spherical compared with the ZnOBtNPs which were larger and hexagonal, while Fourier transform infrared spectroscopy (FTIR) confirmed ZnO-Betaine formation. ZnOBtNPs at 100 mg L − 1 significantly increased the shoot and root length as well as the fresh weight of drought-stressed plants, whereas a higher concentration of ZnOBtNPs determined a stress mitigating response, as shown by a decreased phenolic and flavonoid contents and a reduced oxidative damage coupled with the up-regulation of the antioxidant defence systems. The authors also observed a decrease in free radical scavenging activity and reducing potential in plantlets following NPs application.

The use of NPs as decontaminants is frequent in medicine but has been scarcely applied to plant tissue cultures. Rakhimol et al. formulated casein stabilized and AgNPs, gold (AuNPs) and copper oxide (CuONPs) NPs as decontaminants to eliminate endophytic bacteria from in vitro cultures of Scoparia dulcis , where it is a major constraint. The synthesized AgNPs and AuNPs were spherical shape with an average diameter of 13.5 nm and 3.5 nm, respectively, while CuONPs were spindle shaped with an average thickness of 25 nm. First, the authors isolated and identified the bacterial endophytes of Scoparia dulcis through 16 S rRNA sequencing. Then, dose-response analyses revealed that the Minimum Inhibitory Concentration of AgNPs, AuNPs and CuONPs against all endophytic bacterial contaminants was, respectively, of 0.125, 0.25 and 0.25 mg mL − 1 while the Minimum Bactericidal Concentration for all was 1 mg mL − 1 . Hence, all three AgNPs, AuNPs and CuONPs proved to be effective and lethal against all the isolated bacterial endophytes.

In their work with the quince rootstock QA, Farhadi et al. compared rice husk-derived biogenic silica NPs (SiO 2 NPs) and ZnONPs as additives to improve the growth and proliferation of shoot cultures during a 35-day period. They found that in vitro shoots treated with 1 mg L¯¹ SiO 2 NPs had the highest number of axillary shoots, while plantlets regenerated from media with 2.5 mg L¯¹ ZnONPs exhibited the highest shoot length and number of leaves.

Sharma et al. assessed the use of SiO 2 NPs as elicitors to increase the production of rebaudioside-A (reb-A) by plants of Stevia rebaudiana micropropagated on solid and liquid cultures. Although the authors could not find any clear uniform pattern for all the parameters examined with the different treatments tested, they could observe that various morphological traits (shoot number, shoot length, node number, leaf area and fresh weight) were higher in liquid than in solid cultures and, this, irrespective of the SiO 2 NPs treatment applied. Conversely, solid cultures had a higher chlorophyll and carotenoid content than liquid cultures, the same as for the antioxidant activity, indicative of higher stress for shoots cultured on solid medium where plants also exhibited a significantly higher content of reb-A than those in liquid medium. Moreover, in the solid medium the reb-A content increased further in presence of SiO 2 NPs, while the reverse occurred in liquid medium. These results suggest that the mechanism of uptake and action of SiO 2 NPs in solid and liquid medium is likely to differ.

Using cobalt NPs (CoNPs) in the medium, Van The Vinh et al. assessed the stem elongation and competence for somatic embryogenesis as also the subsequent in vivo growth and flowering of tuberous begonias ( Begonia x tuberhybrida Voss) grown under different light sources (fluorescent lamps - FL, blue LED, red LED, and blue to red LED ratio). After 60 days of culture, shoots cultured under red LEDs were longer, had more internodes per shoot, and larger fresh and dry weights compared to those kept under the other light sources. On the other hand, cultures under red LED exhibited higher somatic embryogenesis, with a larger number of somatic embryos, and a higher percentage of with torpedo-shaped and cotyledonary somatic embryos compared to those under other light sources. Culture of such cotyledonary somatic embryos on a medium containing 0.0465 µg L − 1 CoNPs enhanced plantlet growth, acclimatization, and flowering of plantlets in the greenhouse.

Adabavazeh et al. studied the elicitation of secondary metabolites production from hairy roots of Calotropis procera by supplementing cultures with various concentrations of synthesized Fe 3 O 4 NPs and salicylic acid (SA) to improve their growth and productivity. The addition of Fe 3 O 4 NPs to leaf explant-derived hairy roots determined an increase of growth, soluble sugars, total proteins, and antioxidant enzymes and reduced H 2 O 2 and MDA levels. This effect was significantly greater for hairy roots treated with both Fe 3 O 4 NPs and SA together, than in those exposed to the elicitors individually. Such transformed hairy roots of C. procera had a significantly larger production of essential oil than the intact plant, especially when supplemented with Fe 3 O 4 NPs and SA.

In a separate study of the potential of NPs to improve the production of secondary metabolites of medicinal interest, Ambreen et al. examined the effects of Carbon nanotubes (CNTs) on adventitious roots of Nigella sativa . They revealed that the application of CNTs at 5.0 to 20.0 mg L − 1 significantly enhanced the number of roots induced and their fresh biomass on solid medium. Subsequent experiments using shaken liquid cultures showed that a 4-hour pre-treatment with 10.0 mg L − 1 CNTs permitted the highest root proliferation. Similarly, 2-hour and 4-hour pretreatments resulted in a higher total phenolic and flavonoid content in the adventitious roots than an 8-hour pre-treatment, with optimum results for the 4-hour pretreatment with 25.0 mg L − 1 CNTs. In addition, the DPPH antioxidant activity increased while Phenyl alanine ammonia lyase (PAL) activity decreased with higher CNT concentrations and longer pretreatment durations. In any case, the adventitious roots of N. sativa treated with 5.0 mg L − 1 CNTs exhibited elevated levels of α-thujene, β-pinene, d-limonene, p-cymene, α-terpineol, carvone, and β-Elemene, coupled with significant levels of thymoquinone, thymol (6.4%), and carvacrol (2.3%).

Finally, Allah et al. examined the effects of Chitosan NPs on the growth and genetic transformation of Phoenix dactylifera . Cultures of three commercial date palm varieties were transformed with the AT1G12660 “ Thio-60 ” gene to introduce resistance to fungus infection. Chitosan NPs were efficient in favouring the genetic transformation in all three varieties, as verified by PCR, and the subsequent inoculation of the transgenic plants produced with Fusarium oxysporum showed that they had become resistant after their transformation with the Thio-60 thionin gene.

The constant growth of population added to climate change have led the UN to propose a series of Sustainable Development Goals which will require the development and exploitation of novel approaches and techniques for crop production, but also the optimisation of existing ones. In this context, the advent of nanotechnology provides a range of novel strategies to improve not only seed germination, but also plant growth, associated with a better tolerance to stress and the potential to improve the production of secondary metabolites of medicinal interest. It is precisely in this latter area that NPs have been most frequently studied, as elicitors to produce secondary metabolites. Rather surprisingly, though, this emerging area of research has not yet reached its climax and, currently, its application in food and agriculture remain scarce. Furthermore, much research input is still required about the beneficial and adverse effects of NPs and the evaluation of their hormetic effect on plant development (growth + differentiation) before this technology may be widely applied for several biotechnological applications and also become an innovative option for sustainable agriculture, used as nanofertilizers, nanopesticides, nanosensors, and agri-food agents.

This SI addressed most of these aspects of nanotechnology applied to in vitro plant tissue cultures and, as such, should be of appeal to the large readership of PCTOC.

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Ochatt, S., Abdollahi, M.R., Akin, M. et al. Application of nanoparticles in plant tissue cultures: minuscule size but huge effects. Plant Cell Tiss Organ Cult 155 , 323–326 (2023). https://doi.org/10.1007/s11240-023-02614-3

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Accepted : 31 October 2023

Published : 16 November 2023

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DOI : https://doi.org/10.1007/s11240-023-02614-3

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Role of Plant Tissue Culture in Agricultural Research and Production

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A special issue of Agronomy (ISSN 2073-4395). This special issue belongs to the section " Agricultural Biosystem and Biological Engineering ".

Deadline for manuscript submissions: closed (31 March 2021) | Viewed by 46146

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Dear Colleagues,

Plant tissue culture plays an important role in the field of fundamental research, conservation, and production. Studying plant morphogenesis and plant physiology requires the ability to grow plants in vitro and plant tissue culture techniques provide the best way to accomplish this. Due to the changing climate, preserving plant biodiversity for future crop security and vegetation is of utmost importance. Techniques such as ex-situ conservation are effective in maintaining plant biodiversity. In vitro technologies allow for the optimization and production of plants that can be used for ex-situ conservation. Furthermore, plant propagation by tissue-culture offers an excellent commercial prospect for the industry engaged in the production of ornamental, vegetable, and fruit plants, where the value of the products is high. The micropropagation technique has reportedly been successful in more than 100 species of plants. However, the development of new technologies and protocols that can be effective at the commercial scale is still needed.

This Special Issue is focused on the “Role of Plant Tissue Culture in Agricultural Research and Production.” This will entail novel research studies and reviews focusing on all related topics including plant morphogenesis, plant growth and development, mass propagation, ex-situ conservation, fruit trees, ornamentals, medicinal plants, cannabis, plant tissue culture lab, light conditions, etc.

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• Plant morphogenesis
• plant growth and development
• mass propagation
• ex-situ conservation
• fruit trees
• ornamentals
• medicinal plants
• plant tissue culture lab
• light conditions

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In vitro propagation of Bergenia stracheyi: an alternative approach for higher production of valuable bioactive compounds

• Amin, Awzia
• Sharma, Nancy
• Sultan, Phalisteen
• Gandhi, Sumit G.
• Srinivas, Kota
• Hassan, Qazi Parvaiz
• Ahmed, Zabeer

Bergenia stracheyi, commonly known as `Pashanbheda' or Zakhm-e-Hayat, is a perennial herb that has been recognized for its diverse medicinal properties. The over-exploitation of B. stracheyi has threatened this species. This research aimed to develop a robust tissue culture protocol that can be utilized for rapid micropropagation of B. stracheyi. This protocol is crucial for ensuring the sustainable production of this valuable plant species and preventing the depletion of its natural populations. This study successfully demonstrated an efficient in vitro regeneration protocol by using leaf explants. For shoot induction and multiplication, MS media were supplemented with BAP (6- benzyl amino purine), IAA (indole-3-acetic acid) and NAA (1-naphthalene acetic acid). The most positive response for callus induction was observed on MS media supplemented with BAP (1.5 mg/L) + NAA (1 mg/L). The greatest number of shoots was observed on solid MS media supplemented with BAP (2 mg/L) + IAA (2 mg/L). The maximum shoot response was obtained on MS media supplemented with BAP (2.5 mg/L). The maximum number of roots was achieved on solid MS media without growth regulators. Rooted plantlets were successfully acclimatized in pots and then transferred to an open field. An analytical method using high-performance liquid chromatography and thin layer chromatography was developed for the identification and quantification of two marker compounds (bergenin and gallic acid) in tissue culture extracts as well as in wild samples. The highest contents of bergenin (4.64 mg/mL) and gallic acid (4.29 mg/mL) were found in the methonolic shoot extracts supplemented with BAP (2.0 mg/mL), which were comparable to the amounts present in the rhizomes of wild B. stracheyi (5.54 mg/mL and 3.62 mg/mL, respectively). The integral combination of tissue culture and analytical techniques could serve as a baseline for understanding the important biosynthetic pathways of completely novel, complex and bioactive metabolites of B. stracheyi.

• Conservation;
• Endemic medicinal plants;
• Plant growth regulators;
• Secondary metabolites