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Tissue Culture—A Sustainable Approach to Explore Plant Stresses

Akila wijerathna-yapa.

1 ARC Centre of Excellence for Plant Success in Nature and Agriculture, The University of Queensland, St Lucia, QLD 4072, Australia

2 School of Biological Sciences, The University of Queensland, St Lucia, QLD 4072, Australia

Jayeni Hiti-Bandaralage

3 Centre for Horticultural Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, Brisbane, QLD 4068, Australia

4 J&S Plant Biotech, P.O. Box 4700, Sunshine Coast MC, QLD 4560, Australia

Associated Data

Not applicable.

Plants are constantly faced with biotic or abiotic stress, which affects their growth and development. Yield reduction due to biotic and abiotic stresses on economically important crop species causes substantial economic loss at a global level. Breeding for stress tolerance to create elite and superior genotypes has been a common practice for many decades, and plant tissue culture can be an efficient and cost-effective method. Tissue culture is a valuable tool to develop stress tolerance, screen stress tolerance, and elucidate physiological and biochemical changes during stress. In vitro selection carried out under controlled environment conditions in confined spaces is highly effective and cheaper to maintain. This review emphasizes the relevance of plant tissue culture for screening major abiotic stresses, drought, and salinity, and the development of disease resistance. Further emphasis is given to screening metal hyperaccumulators and transgenic technological applications for stress tolerance.

1. Introduction

Plant tissue culture satisfies large-scale plant propagation needs and is an essential tool facilitating other biotechnology applications in plant improvement space [ 1 , 2 , 3 ]. In addition, its importance as a tool and direct application in fundamental studies relating to plant biology, biochemistry, and molecular biology is well recognized. Today, the world population has reached an alarming 8 billion people. Providing food and products, of which the majority is plant-based, has become one of the biggest challenges in front of the human race in this era, while the unprecedented climate change challenges are bigger than ever before [ 4 , 5 , 6 ]. Various climatic changes as a result of global warming greatly influence agriculture systems around the globe. This includes severe environmental pressures, drought, extreme heat or cold climate, floods, salinity, and exposure to toxic compounds [ 4 ]. Human activities in the current production-based economy also contribute toward changes in soil and environmental conditions due to the accumulation of toxins and chemical elutes released to the environment in production processors [ 7 ]. The flip side of this is the reduction in agricultural production, flagging food security due to the decrease in plant growth and development due to various environmental stress factors and the decline and scarcity of suitable agricultural land.

The focus on plant stress-related research has gained substantial momentum over the last four decades, especially on the impact of stress factors such as water deficiency, extreme temperatures, salinity, exposure to toxic compounds, inadequate or extreme radiation, plant infection with pathogens, and pest outbreaks [ 8 , 9 ]. Screening plants for various biotic and abiotic stress conditions is vital in breeding and selecting elite varieties. Most plant screening trials for stresses are conducted under field conditions, yet very challenging to manage, both physically and economically, and subject to various risks due to dynamic external environments.

Plant tissue culture provides an effective, efficient, and comparatively economical platform to screen plants for biotic and abiotic stresses. Plant cell and tissue culture, also known as in vitro culture, is based on the cell theory of Schwann and Schleiden (1838) and the ideas of Gottlieb Haberlandt at the beginning of the 20th century [ 10 ]. In vitro plant tissue culture is based on cells’ “totipotency” or “total potential”. Theoretically, every cell can become a fully grown plant when provided with suitable conditions. Totipotency has been better described as the ability of any fully functional components of plants to undergo dedifferentiation and redifferentiate to form an organized tissue, structure, and eventually a whole organism [ 11 , 12 ]. Based on this phenomenon, whole plants develop when plant cells or tissues are provided with specific nutrients and optimal growth conditions under an in vitro sterile environment. In vitro culture of plants under defined culture media composition with the ability to impose variables with no external environmental influence while maintaining control environment parameters offers the opportunity for more efficient screening for desirable characteristics. This is also applied to test tolerance to selective agents such as toxins and antibiotics. Utilization of in vitro selection can considerably shorten the time and cost of the selection process under selection pressure with minimal environmental interaction. Conducting in vitro screening for stress will not replace but complement field selection resulting in better insight and outcome. Moreover, it can be used as an early evaluation platform to understand and provide direction with justification for the further need for field evaluation. As with any technique or procedure, in vitro screening for stress tolerance has its challenges. The biggest challenge is the requirement for reliable and established tissue culture protocols for specific plant species. Another issue is the lack of correlation between the mechanisms of tolerance operating at the cellular or tissue level in cultured cells to those of whole plants. Epigenetic adaptation can also interfere with the results as non-tolerant cells may have an epigenetic adaptation during in vitro culture process [ 13 , 14 , 15 ]. This type of epigenetic adaptation can be overcome using a short-term or one-step in vitro selection process.

Studies on plant biodiversity, especially crop species, have increased tremendously over the years in search of better and more resilient crops despite breeding attempts. The research focused on plant improvement and selection has taken a massive hype for searching elite species for new and better chemicals, disease resistance, productivity, and consumer preference. This review elaborates on how in vitro culture is utilized to screen plant species for different biotic and abiotic stress evaluation studies with examples of a broad variety of crop species. The focus is on in vitro screening for the main stresses: drought, salinity, and disease resistance. This review also highlights other uses of in vitro screening for the identification of metal accumulators and stress-tolerant transgenic plant development with further discussion on the pros and cons of specific studies and the effectiveness of the in vitro culture tools utilized as fast and cost-effective alternative in plant screening.

2. In Vitro Screening of Drought Tolerance

Drought affects plant growth and development, reflecting plants’ productivity [ 16 , 17 ]. Drought stress impacts crop performance at several phases in the plant’s life cycle, from emergence to maturity, including seed germination, vegetative growth, and reproductive development, ultimately affecting the quality and quantity of the harvest. Drought, especially in arid and semi-arid regions, causes significant agricultural losses [ 18 ]. Under drought stress, several molecular, biochemical, physiological, morphological, and ecological characteristics and processes of plants are affected due to the triggering of stress-responsive factors [ 19 ]. As a result, the productivity and quality of plants diminish in water-deficient situations. Growth stages, age, plant species, drought intensity, and duration of drought period are the primary determinants for the responses elicited by the plant in response to drought stimuli.

Plants process various mechanisms to adopt, tolerate, or resist drought conditions. However, the ability and the level of tolerance/resistance differ among and within plant species, especially when genetic variability exists due to outcrossing or natural mutation [ 16 , 19 , 20 , 21 ]. Drought responses are governed by activating signal transduction pathways linked with molecular networks to elicit survival or adaptation mechanisms [ 20 ]. In general, drought causes a reduction in soil moisture and results in reduced water potential in root cells [ 17 ]. For many decades, breeding and selection for drought tolerance or resistance have been major research areas for many crop species i.e., rice, maize, and sorghum [ 22 , 23 , 24 , 25 , 26 , 27 ]. However, screening for drought in field conditions requires a substantial amount of resources (land, labor, and energy), which is costly, and is associated with challenges in relying on nature for stable environmental conditions to efficiently and effectively replicate data in expressing exact genotype [ 21 ].

In vitro applications for drought screening can be a smart and easy method compared to field studies. The strategy to apply drought conditions in vitro is to impose similar conditions created at cellular levels when plants are subjected to drought in the field environment. Under abiotic stress, plants accumulate solutes or osmolytes within the cell due to less water availability. Therefore, high molecular weight solutes such as sucrose, sorbitol, mannitol, and poly-ethylene glycol (PEG) are suitable candidates to impose physiological drought under in vitro conditions [ 28 , 29 , 30 , 31 , 32 ]. In addition, these osmolytes stabilize proteins and cell membranes’ structure during dehydration stress conditions [ 33 , 34 ].

Contrary to drought stress, which induces osmotic stress in plants, accumulating these chemicals reduces osmotic potential, preserving cellular turgor and enhancing water absorption [ 20 , 35 ]. Moreover, they play a crucial function in protecting plant cells from oxidative stress by removing reactive oxygen species [ 20 , 32 , 35 ]. It has been shown that sucrose accumulates in plant tissues under drought stress [ 36 , 37 , 38 ]. PEG, sucrose, mannitol, and sorbitol have been the main chemicals for imposing osmotic pressure in vitro. PEG has reportedly been used to impose physiological drought in plants [ 28 , 29 , 39 , 40 ]. This high molecular weight chemical is an inert, non-penetrating osmoticum that decreases the water potential of nutritional solutions without being taken up by the plant or phytotoxic. Since PEG does not reach the apoplast, it drives water from the cell wall and interior. Therefore, PEG solutions resemble dry soil more closely than low molecular weight chemicals, which permeate the cell wall with solute. PEG is not used in the cellular metabolism of plants, but it does induce water stress by lowering the water potential of nutrient solutions, hence inhibiting plant development in vitro [ 28 , 39 , 41 ]. It has no harmful or toxic effects on the plant; nonetheless, it restricts plant development by lowering the water potential of the culture medium, such as water deficit soil, preventing cultured explants from absorbing water [ 42 ]. Mannitol and sorbitol have been usually used as osmotic pressure regulators in plant in vitro cultures while utilized as a carbon source [ 43 ]. Several studies have applied PEG, mannitol, and sorbitol for in vitro drought screening studies ( Table 1 ).

Application of different chemicals of in vitro screening for drought tolerance.

3. In Vitro Screening of Salinity Tolerance

Salinity in soil and water is the most critical constraint that affects plant growth and development [ 58 , 59 ]. Due to osmotic or ionic stress or nutritional imbalance, salinity stress has a deleterious effect on plant development [ 60 , 61 , 62 ]. Arid and semi-arid environments are characterized by accumulating large quantities of salts in the soil [ 63 ]. Although many remedial and management procedures are utilized to make salt-affected soils suitable for agriculture, they are exceedingly costly and do not offer lasting answers to the salinity problem. Therefore, salinity stress has gained considerable traction over the past few decades due to the vast experimental evidence from what has occurred in nature regarding the evolution of highly salt-tolerant ecotypes of various plant species [ 64 , 65 , 66 ], as well as the remarkable progress made in improving various agronomic traits through artificial selection [ 67 ]. Plant tissue culture is the most efficient method for enhancing and producing salt tolerance in plants. By utilizing plant cell and tissue culture, it is possible to focus on the physiological and biochemical processes crucial to the cell and contribute to the alterations brought about by salt stress. Using two in vitro culture methods, salt-tolerant plants have been obtained through cell and tissue culture procedures. The first method involves selecting mutant cell lines from cultivated cells, followed by plant regeneration using these cells (somaclones). The second method is the in vitro screening of plant germplasm for salt tolerance, which has been successfully used in durum wheat [ 68 ]. Doubled haploid lines generated from pollen culture of salt-tolerant F1 hybrid parents have the potential to enhance salt tolerance [ 69 , 70 ]. Somaclonal variation and in vitro-induced mutagenesis can create variability from which crop plants can be improved. Examples of other in vitro selections for increased resistance to salt stresses are shown in Table 2 . Enhancing resistance to both hyper-osmotic stress and ion toxicity may also be accomplished by molecular breeding of salt-tolerant plants employing molecular markers or genetic engineering.

In vitro selection for increased resistance to salt stresses.

4. In Vitro Screening of Disease Resistance

Plant diseases cause substantial revenue loss in agriculture due to lost or poor performing plants when infected. Thus, breeding and selection for disease resistance are at the forefront of crop science. Various air, soil, and water-borne fungal, bacterial, viral, and mycoplasma diseases affect commercial crops, especially in monoculture and chemically fertilized environments. Therefore, studying and screening for disease tolerance and resistance are routine operations. Often such investigations require specialized conditions and highly controlled setups to minimize the risk of an unintentional spread of diseases to the outside environment causing disease outbreaks for major agricultural crops. In vitro selection using pathogenesis-related proteins, antifungal peptides, or phytoalexin production can help select elite-resistant varieties. This method is simpler and cheaper than generating plants through transgenic technology, which is costly, time-consuming, and more challenging to commercialize due to policy and social acceptance barriers. Exposing organogenic or embryogenic calli, shoots, somatic embryos, or cell suspensions to pathogen toxins, culture filtrate, or the direct pathogen can effectively screen plant samples for pathogen resistance in vitro. In Table 3 , such in vitro screening studies are listed for various crop species.

In vitro selection for increased resistance to biotic stresses.

Some research suggests that rather than the success of screening, somaclonal variation occurring during the tissue culture process is a probable factor in disease-resistant behavoir [ 91 , 92 , 93 , 94 ]. A combination of a chance mutation in vitro with in vitro selection pressure appears to delay and confound the later examination of plants produced by these methods. In such an attempt by Vos et al. (2000), they grew tens of thousands of standard seedlings in culture and screened in vitro for resistance to guava wilt disease [ 95 ]. This is the most impressive example of such in vitro screening study for disease resistance. This accelerated the discovery of potentially resistant plants, saving the South African guava industry.

5. In Vitro Screening of Metal Hyperaccumulators

Phytoremediation is a novel and cost-effective method for removing hazardous heavy metals (Pb, Cd, Cu, Zn, etc.) and organic contaminants from water and soil [ 7 ]. There are now accessible biotechnologies for better comprehending plants’ mechanism of heavy metal absorption and examining their potential for remediation enhancement [ 96 ]. On the other hand, metal accumulators are highly beneficial and trendy as food supplements or for recouping rare and expensive metal elements for cosmetics and other uses [ 97 ]. When studying the tolerance of plant cells to hazardous substances, in vitro cultures provide several advantages [ 98 ]. In this regard, in vitro screening is a preliminary technique for assessing woody plant materials since it reduces the time required for growth and treatment and the amount of space necessary for the tests. Since it is conducted under controlled conditions, plant tissue culture is one of the most dependable procedures used in fundamental research to establish the metabolic capacity of plants [ 99 ]. Research on phytoremediation typically uses several plant tissue cultures as model plant systems. Some examples of these cultures are calli, cell suspensions, and hairy roots. When it comes to research on the inherent metabolic capacities of plant cells and their ability to tolerate toxicity, in vitro cultures provide several benefits to the otherwise unavailable experimentation process. In the quest for fundamental information about plants, the capacity to determine the specific contributions that plant cells make to the process of pollutant absorption and detoxification in the absence of interference by microbes is of special value. However, the final objective of such studies is to develop a realistic phytoremediation technology. In that case, it is necessary to understand the inherent limitations in using in vitro cultures as a representative of entire plants in the field. It is highly likely that the bioavailability of contaminants and the processes of pollutant uptake and metabolite distribution will be significantly different in the two systems. This can lead to qualitative and quantitative differences in metabolic profiles and tolerance characteristics. In order to gain complete understanding or to identify an effective species in phytoremediation through chemical accumulation, it is necessary to use intact rooted plantlets in tissue culture conditions for screening studies. However, several studies have shown that plant tissue culture is a handy tool in the field of phytoremediation surveys ( Table 4 ). The findings obtained from tissue cultures may be utilized to make predictions regarding the reactions of plants to environmental toxins, as well as to enhance the design of future traditional whole plant tests, which in turn helps to lower their overall costs.

In vitro selection for increased resistance to hyperaccumulators.

6. Stress Tolerance through Transgenic Technology

Genetic modification technology for agriculturally important plant species has achieved major advances in the last decade. The development of transgenic plants with desirable characteristics, such as tolerance to biotic and abiotic stress, is a reality. Plant characteristics are altered much faster than ever by utilizing a wide variety of approaches through gene transfer and gene editing [ 2 , 4 , 117 ]. The technique for tissue regeneration through tissue culture is a prerequisite in such processes, and it is vital.

Genetic transformation has been proposed for several decades as a quick way to modify the morphological characteristics of an organism. Plant genetic transformation techniques can be classified as direct and vector-based, introducing transgenic DNA to the host organism. Direct genetic transformation refers to the direct introduction of transgenic DNA to a plant cell. The most used techniques are biolistics and biological vectors that use Agrobacterium tumefaciens -mediated transformation [ 2 ].

Genetic transformation through biolistics can transform any totipotent plant cell, from which cell lines, tissues, or whole plants can be created. However, it has the disadvantage that the transformation can be transient and generate chimeric plants (non-transformed cells within the plant), in addition to the requirement of expensive equipment and low transformation efficiency [ 118 ]. On the other hand, the most studied and used vector-based transformation method is through infection of Agrobacterium tumefaciens , a natural plant pathogenic bacterium capable of incorporating a DNA region (Ti plasmid of the Agrobacterium tumefaciens ) into the plant genome. This system does not require specialized equipment and thus is inexpensive, and the number of transformation events per cell is limited. However, limitations exist due to plant regeneration challenges from the transformed callus cells [ 119 , 120 , 121 ].

The methods of introducing foreign DNA or changing plant genomes have been updated for the use of more defined transformation systems such as the “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated protein 9 (Cas9) systems”. This system is powerful to allow specific genetic edits to change genomes according to the need. The CRISPR/Cas9 system is based on the immune system of bacteria adapted to eukaryotic systems, including plants [ 122 , 123 ], whose principle is the RNA–DNA interaction to search for the sequence genomics [ 124 , 125 ].

Not all plant tissue types and species are conducive to the above transformation methods. An explant can be a variety of tissues, depending on the particular plant species and its regenerative ability. Table 5 below lists several studies conducted to achieve abiotic and biotic resistance through genetic transformation. Identification of a suitable tissue culture approach to maximize the transformation efficiency with higher regeneration of transformed cells is critical. Different approaches are illustrated in Figure 1 .

Different techniques in tissue culture for plant regeneration can be utilized for the selection and genetic transformation of plants. Starting from explants under selection mediums, direct organogenesis can be achieved (A and B) or indirect organogenesis (C and D) through an intermediate callus phase. Further, callus can be used to form intact plantlets through an embryonic pathway or in suspension culture directly or via protoplast culture techniques in genetic transformation attempts.

Examples of attempts to improve abiotic stress tolerance in crop plants through genetic transformation.

Different combinations of culture type and transformation protocol are used depending on the plant species and cultivar. In some species, various culture types and regeneration methods can be used, which enables a wide variety of transformation protocols to be utilized. However, there is no choice over culture type and regeneration method in other species, limiting the applicable transformation protocols [ 136 ].

Salinity, drought, water logging, heat, frost, and mineral toxicities limit commercial agricultural productivity. Biotechnology can bring in solutions to increase crop productivity. Tissue culture-based in vitro selection and mutagenesis have become a viable and affordable method for stress-tolerant plant development. Current research supports the notion that in vitro screening is an alternative and a support platform for stress tolerance screening for drought, salinity, and chemical toxicity. Further, in vitro culture permits accurate modification and assessment of stress variables, identifying stress-tolerance genes and metabolic pathways. Expanding research into this area of science will ensure more efficient and effective screening methodologies for the future of sustainable agriculture.

Acknowledgments

A.W.-Y. thanks the Australian Research Council Centre of Excellence for Plant Success in Nature and Agriculture.

Funding Statement

This research received no external funding.

Author Contributions

A.W.-Y. and J.H.-B. conceptualized the idea of the review. A.W.-Y. prepared the first draft. J.H.-B. reviewed and edited the manuscript and produced the figure. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Informed consent statement, data availability statement, conflicts of interest.

The authors declare that there is no conflict of interest.

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MINI REVIEW article

The role of silicon in plant tissue culture.

• Department of Molecular Biotechnology, Konkuk University, Seoul, South Korea

Growth and morphogenesis of in vitro cultures of plant cells, tissues, and organs are greatly influenced by the composition of the culture medium. Mineral nutrients are necessary for the growth and development of plants. Several morpho-physiological disorders such as hooked leaves, hyperhydricity, fasciation, and shoot tip necrosis are often associated with the concentration of inorganic nutrient in the tissue culture medium. Silicon (Si) is the most abundant mineral element in the soil. The application of Si has been demonstrated to be beneficial for growth, development and yield of various plants and to alleviate various stresses including nutrient imbalance. Addition of Si to the tissue culture medium improves organogenesis, embryogenesis, growth traits, morphological, anatomical, and physiological characteristics of leaves, enhances tolerance to low temperature and salinity, protects cells and against metal toxicity, prevents oxidative phenolic browning and reduces the incidence of hyperhydricity in various plants. Therefore, Si possesses considerable potential for application in a wide range of plant tissue culture studies such as cryopreservation, organogenesis, micropropagation, somatic embryogenesis and secondary metabolites production.

Introduction

Plant tissue culture is a collection of experimental procedures for aseptic culture of isolated plant cells, tissues or organs on nutrient media under controlled environmental conditions. Growth and morphogenesis of in vitro cultures of plant cells, tissues and organs are greatly influenced by the composition of the culture medium. The composition of a culture medium has often been modified to stimulate the growth of particular plant material. In general, plant tissue culture medium composed of inorganic nutrients, organic supplements, carbon source, plant growth regulators and a solidifying agent. Mineral nutrients are necessary for growth and development of plants. The optimization of inorganic nutrients in the culture medium improves growth and morphogenesis of plant cells, tissues and organs in vitro . Several physiological disorders such as hooked leaves, hyperhydricity, fasciation and shoot tip necrosis are often associated with the concentration of inorganic nutrients in the culture medium ( Reed et al., 2013 ).

Silicon (Si) is the most abundant mineral element in the soil ( Epstein, 1999 ). Numerous studies have shown that Si treatment improves the growth and yield of various plants, particularly when they are subjected to both abiotic and biotic stresses ( Ma, 2004 ). Several researchers have reviewed the role Si on plant tolerance to abiotic ( Balakhnina and Borkowska, 2013 ; Zhu and Gong, 2014 ) and biotic stresses ( Van Bockhaven et al., 2013 ). The availability of Si in hydroponic and substrate plant production system is restricted. Addition of Si to the nutrient solution or soilless substrate enhanced growth traits, yield and quality of several crops ( Voogt and Sonneveld, 2001 ). The promoting effects of Si on plants might be due to increasing nutrient uptake and photosynthetic activity. Though Si is a ubiquitous contaminant, the use of silicon-free containers and double distilled water restricts its availability. Furthermore, Si has not been included in any commercial tissue culture media formulation. The inclusion of Si to the culture medium improved the morphogenetic potential of plant cells, tissues and organs. Several studies have shown that the inclusion of Si to the tissue culture medium enhances callus growth, shoot regeneration, and root induction and stimulates somatic embryogenesis, and improve morphological, anatomical and physiological characteristics of plantlets. In addition, Si treatment prolongs the longevity of calli and organs with a potential for plant regeneration. The inclusion of Si to the tissue culture medium also enhances tolerance to low temperature, metal toxicity and salinity. Si enhancing tolerance of plants to various stresses by altering activity of antioxidant enzymes, cation binding capacity of the cell walls, endogenous plant hormone level, increasing production of chitinase, glucanse, lignin, phenolics, and phytoalexins, nutrient uptake, improving strength of cell and plant, maintaining the structure of stomata, relative water content, and reducing uptake of heavy metals. This review concentrates the potential roles of Si in plant tissue culture.

Role of SI in Plant Tissue Culture

Organogenesis and somatic embryogenesis.

Islam et al. (2005) investigated the effect of calcium silicate (CaSiO 3 ) on callus induction and plant regeneration from mature seed explants of rice ‘Kalizira’, ‘Lucky’, and ‘Pajam’. The highest frequency of callus induction is achieved on Murashige and Skoog (MS) medium containing CaSiO 3 . However, plant or root regeneration potential of rice calli is cultivar depended. Similarly, effects of Si on plant or root development depend on reed ( Phragmites australis ) genotype used for callus induction ( Mathe et al., 2012 ). Addition of Si as sodium silicate (Na 2 SiO 3 ) to the modified MS medium promotes the growth of calli obtained from stem nodal and root explants of P. australis while its effect on somatic embryogenesis is explants dependent: it stimulates embryogenesis of root calli, but it does not influence this process in stem nodal calli. Soares et al. (2011) evaluated the effect of Si source [potassium silicate (K 2 SiO 3 ) and Na 2 SiO 3 ] on shoot multiplication of Cattleya loddigesii . The highest number of shoots is observed on the modified Knudson C medium containing 5.0 mg L -1 K 2 SiO 3 . In Ajuga multiflora , addition of Si to MS medium containing 2iP and IAA, enhanced adventitious shoot regeneration (about threefold) by increasing the activity of antioxidant enzymes such as SOD, POD, APX, and CAT ( Sivanesan and Jeong, 2014 ). In addition, the authors observed the Si accumulation in leaves of plants developed in the culture medium with Si, but not in plants developed in the medium without Si by wavelength dispersive X -ray analysis. These studies indicate that the effect of Si on morphogenetic potential of in vitro plant cultures depends on plant species, genotype and concentration of Si in the culture medium. Still further studies are required to better understand the biochemical and molecular mechanism of Si on organogenesis and somatic embryogenesis.

Growth and Development

The application of Si has been reported to enhance the growth and development of various plants. Zhou (1995) observed silica bodies in leaf tissues of Phalaenopsis hybrid plantlets grown in Vacin and Went medium supplemented with CaSiO 3 . Addition of CaSiO 3 also increased the leaf length. Soares et al. (2011) reported that the addition of 5.0 mg L -1 K 2 SiO 3 and 20.0 mg L -1 Na 2 SiO 3 to the modified Knudson C medium increased the number of roots and length of aerial part and root in seedlings of C. loddigesii . Subsequently the same group investigated the effect of GA 3 and Na 2 SiO 3 on growth and development of C. loddigesii ( Soares et al., 2013 ). It was reported that the combination of GA 3 and Na 2 SiO 3 increased the number of leaves and roots than GA 3 alone. The optimal concentration of Si varies within the same plant species and or genotype. The inclusion of CaSiO 3 at 0.5 and 2.0 mg L -1 to the MS medium stimulates the growth of native ( Brassavolva perrinii ) and hybrid ( Laelia cattleya ‘Culminant Tuilerie’ × L. cattleya ‘Sons Atout Rotunda’) × Brassolaelia cattleya ‘Startifire Moon Beach’) orchid plants, respectively ( Soares et al., 2012 ). Lim et al. (2012) also reported that the effect of Si (K 2 SiO 3 ) on the growth traits of begonia ‘Super Olympia Red’ and ‘Super Olympia Rose’ and pansy ‘Matrix White Blotch’ and ‘Matrix Yellow Blotch’ are mainly dependent on the cultivars. Braga et al. (2009) investigated the effect of different Si sources such as CaSiO 3 , K 2 SiO 3 , and Na 2 SiO 3 on the growth and anatomical characteristics of strawberry ‘Oso Grande’ seedlings. The fresh and dry weight of seedlings increased in MS medium containing 1.0 g L -1 Na 2 SiO 3 . Seedlings of banana ‘Maca’ cultured in the medium supplemented with CaSiO 3 increased the chlorophyll content, whereas those cultured in the medium containing Na 2 SiO 3 increased length, fresh and dry weight of shoots ( Asmar et al., 2011 ).

The morphological and anatomical characteristics of in vitro -grown plantlets are different from the field-grown seedlings. Si inclusion to the rooting medium increased leaf tissue thickness and epicuticular wax deposition in banana ( Asmar et al., 2013a ) and strawberry ( Braga et al., 2009 ) plantlets. Luz et al. (2012) reported that supplementation of CaSiO 3 , K 2 SiO 3 , or Na 2 SiO 3 to the rooting medium improved leaf anatomy of banana ‘Maca’ plantlets. The inclusion of CaSiO 3 to the culture medium also increased photosynthetic rate and chlorophyll content of banana plantlets ( Asmar et al., 2013b ). In strawberry, light and electron microscopic analysis showed deformation in chlorenchyma and the epidermis of leaves from plantlets grown in the culture medium devoid of Si ( Soares et al., 2012 ). Recently, He et al. (2013) confirmed the deposition of Si within the cell walls of in vitro -cultured rice cells. Si improves the structural stability of cell walls during cell elongation and division and thereby maintained cell shape, which may be important for the function and survival of cells (Table 1 ).

TABLE 1. Role of Si in plant tissue culture.

Ziv (2010) investigated the effect of silicon on hyperhydricity in Ornithogalum dubium . Addition of Na 2 SiO 3 to MS liquid medium containing BA, NAA and 6% sucrose in bioreactors, significantly reduced induction of hyperhydric shoots, and increased plant firmness and mechanical strength. Si treatment significantly reduced the content of hydrogen peroxide and activity of oxidative reductive enzymes such as APX, ascorbate oxidase and GPX in leaves of the regenerated shoots of O. dubium when compared with the control (Table 1 ). Similarly, addition of Si as K 2 SiO 3 to MS medium reduced the hyperhydricity in Cotoneaster wilsonii by decreasing the content of MDA in the regenerated shoots when compared with the control ( Sivanesan et al., 2011 ). The authors observed the presence of Si in the in non-hyperhydric plants, but not in the hyperhydric leaf samples of C. wilsonii by energy dispersive X -ray analysis. Thus, the problem of hyperhydricity can be reduced by the inclusion of Si to both liquid and solid culture medium. Phenolic oxidative tissue browning is one of the bottlenecks in woody plant tissue culture. In guava, tissue browning was completely prevented by sealing the nodal explants cut ends with Si ( Youssef et al., 2010 ) and there was no detrimental effect of Si on the subsequent steps of in vitro propagation. The authors suggested that Si could be used during explants preparation to control phenolic tissue browning in other plants. The morphological, anatomical and physiological characteristics of plantlets can improve in vitro by incorporating Si in the culture medium. However, further studies required to evaluate the effect of different source and concentration of silicon on the growth and development of various plants.

Abiotic Stress Tolerance

Duan et al. (2013) reported that Si enhance cold resistance of Dendrobium moniliforme by increasing the content of free proline, soluble sugar and soluble protein and decreasing MDA content. Si treatment improved the survival rate of grape ‘Kyoho’ and ‘koshusanjaku’ calli under low temperature by preventing browning ( Moriguchi et al., 1988 ). In vitro storage of Coleus hybridus ‘ Jupiter’ and Solanum tuberosum var. Gersa under silicone oil significantly reduced the growth and maintained their regenerative potential ( Radovet et al., 2008 ; Radovet-Salinschi and Cachita-Cosma, 2012 ). These results reveal that Si can be used as cryoprotectant and included in the cryoprotective mixture for minimizing the toxicity of cryoprotectants. The ameliorating effect of Si on salt stress in vitro has been reported in A. multiflora ( Sivanesan and Jeong, 2014 ), Salvia splendens ‘Hot Jazz’ ( Soundararajan et al., 2013 ) and S. tuberosum ( Qing et al., 2005 ). Si alleviates salt stress in plants by limiting NaCl uptake, maintenance of ultrastructure of stomata, improving photosynthetic activity, reducing free proline content and altering the production of antioxidant enzymes ( Qing et al., 2005 ; Soundararajan et al., 2013 ; Sivanesan and Jeong, 2014 ). Prabagar et al. (2011) investigated the effect of Si on aluminium (Al) tolerance in Picea abies suspension cultures. Al toxicity was reduced when the liquid medium was supplemented with Si and the effect was increased at pH 5.0 than pH 4.2. Si supplementation protected P. abies cells and against Al toxicity by reducing the concentration of free Al in the cell wall. Si is also reported to enhance drought tolerance, alleviate lead toxicity and increase resistance to radiation and temperature stresses ( Balakhnina and Borkowska, 2013 ; Zhu and Gong, 2014 ). The molecular mechanisms of Si on stress tolerance are poorly understood. Thus, more studies are needed to find out the role of Si in abiotic tolerance on various plants.

Future Prospects

Recent studies have shown the beneficial effects of Si in plant tissue culture (Table 1 ). However, further studies on a wide variety of plant species are needed to confirm the role of Si in plant tissue culture. In vitro culture is a useful system for studying physiological and biochemical functions of Si in plants at molecular level. Further, in vitro cell suspension culture systems provide an opportunity to study roles of Si at the single cell level. The inclusion of silicone A to Linsmaier and Skoog liquid medium also enhances cell growth and anthocyanins content in cell suspension culture of Perilla frutescens ( Zhong et al., 1992 ). Thus, Si can also be used for the stimulation of secondary metabolites in the plant cell, tissue and organ cultures. We strongly recommend the inclusion of Si as a beneficial nutrient in the tissue culture medium to solve various micropropagation problems, and to increase tissue culture success.

Conflict of Interest Statement

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.

Acknowledgment

This article was supported by the KU Research Professor Program of Konkuk University.

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Keywords : acclimatization, epicuticular wax deposition, hyperhydricity, organogenesis, silicon, stress tolerance

Citation: Sivanesan I and Park SW (2014) The role of silicon in plant tissue culture. Front. Plant Sci. 5 :571. doi: 10.3389/fpls.2014.00571

Received: 01 August 2014; Accepted: 04 October 2014; Published online: 21 October 2014.

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Copyright © 2014 Sivanesan and Park. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Se Won Park, Department of Molecular Biotechnology, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, South Korea e-mail: [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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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Published : 16 November 2023

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

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Sumitomo Rubber studying ‘rubber tree tissue culture' technique

15 May 2024

Joint research in Thailand examines yields from test tube technique compared to conventional grafting 

Hyogo, Japan – Sumitomo Rubber Industries (SRI) has entered a partnership with the Khon Kaen University of Thailand to jointly research ‘rubber tree tissue culture' techniques.

The research aims to advance the productivity of natural rubber by studying mechanisms for yield-improvement of rubber trees, said the Japanese group.

Grafting is generally used to propagate rubber tree seedlings, but the process is “significantly affected” by the stock in terms of growth, disease resistance, and other issues.

With the alternative technique, explained SRI, part of the tissue of a rubber tree is specially isolated and cultured in a test tube.

As part of the project, the partners will compare the growth and leaf shapes of rubber tree seedlings derived from tissue culture with those derived from grafting.

The research team will then analyse plant physiological responses chiefly through the measurement of transpiration and evaluating the differences.

“It has been confirmed that the seedlings deriving from tissue culture grow faster than the seedlings deriving from general grafting in the early stage of planting,” said SRI.

The growth difference between the two can vary by up to two years, according to the Japanese group's statement.

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• 09 May 2024

Cubic millimetre of brain mapped in spectacular detail

• Carissa Wong

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Rendering based on electron-microscope data, showing the positions of neurons in a fragment of the brain cortex. Neurons are coloured according to size. Credit: Google Research & Lichtman Lab (Harvard University). Renderings by D. Berger (Harvard University)

Researchers have mapped a tiny piece of the human brain in astonishing detail. The resulting cell atlas, which was described today in Science 1 and is available online , reveals new patterns of connections between brain cells called neurons, as well as cells that wrap around themselves to form knots, and pairs of neurons that are almost mirror images of each other.

The 3D map covers a volume of about one cubic millimetre, one-millionth of a whole brain, and contains roughly 57,000 cells and 150 million synapses — the connections between neurons. It incorporates a colossal 1.4 petabytes of data. “It’s a little bit humbling,” says Viren Jain, a neuroscientist at Google in Mountain View, California, and a co-author of the paper. “How are we ever going to really come to terms with all this complexity?”

Slivers of brain

The brain fragment was taken from a 45-year-old woman when she underwent surgery to treat her epilepsy. It came from the cortex, a part of the brain involved in learning, problem-solving and processing sensory signals. The sample was immersed in preservatives and stained with heavy metals to make the cells easier to see. Neuroscientist Jeff Lichtman at Harvard University in Cambridge, Massachusetts, and his colleagues then cut the sample into around 5,000 slices — each just 34 nanometres thick — that could be imaged using electron microscopes.

Jain’s team then built artificial-intelligence models that were able to stitch the microscope images together to reconstruct the whole sample in 3D. “I remember this moment, going into the map and looking at one individual synapse from this woman’s brain, and then zooming out into these other millions of pixels,” says Jain. “It felt sort of spiritual.”

A single neuron (white) shown with 5,600 of the axons (blue) that connect to it. The synapses that make these connections are shown in green. Credit: Google Research & Lichtman Lab (Harvard University). Renderings by D. Berger (Harvard University)

When examining the model in detail, the researchers discovered unconventional neurons, including some that made up to 50 connections with each other. “In general, you would find a couple of connections at most between two neurons,” says Jain. Elsewhere, the model showed neurons with tendrils that formed knots around themselves. “Nobody had seen anything like this before,” Jain adds.

The team also found pairs of neurons that were near-perfect mirror images of each other. “We found two groups that would send their dendrites in two different directions, and sometimes there was a kind of mirror symmetry,” Jain says. It is unclear what role these features have in the brain.

Proofreaders needed

The map is so large that most of it has yet to be manually checked, and it could still contain errors created by the process of stitching so many images together. “Hundreds of cells have been ‘proofread’, but that’s obviously a few per cent of the 50,000 cells in there,” says Jain. He hopes that others will help to proofread parts of the map they are interested in. The team plans to produce similar maps of brain samples from other people — but a map of the entire brain is unlikely in the next few decades, he says.

“This paper is really the tour de force creation of a human cortex data set,” says Hongkui Zeng, director of the Allen Institute for Brain Science in Seattle. The vast amount of data that has been made freely accessible will “allow the community to look deeper into the micro-circuitry in the human cortex”, she adds.

Gaining a deeper understanding of how the cortex works could offer clues about how to treat some psychiatric and neurodegenerative diseases. “This map provides unprecedented details that can unveil new rules of neural connections and help to decipher the inner working of the human brain,” says Yongsoo Kim, a neuroscientist at Pennsylvania State University in Hershey.

doi: https://doi.org/10.1038/d41586-024-01387-9

Shapson-Coe, A. et al. Science 384 , eadk4858 (2024).

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