《1. Introduction》

1. Introduction

The field of bioengineering focuses on the application of biological principles to generate economically useful products. Bioengineering is needed for medical devices, diagnostic tools, biocompatible materials, recyclable bioenergy, agricultural engineering, and more. The aim of bioengineering is to rebuild or modify biologic systems in order to generate marketable products in the fields of biotechnology, microbiology, biocatalysis, and others. Tissue engineering is not only related to human (or animal) tissue replacement, but also to plant tissues. Furthermore, the pharmaceutical sciences include engineering technologies to produce chemical drugs and recombinant proteins (i.e., therapeutic antibodies). This novel field has been termed pharmaceutical engineering.

Since time immemorial, humans have used plant products as sources for pharmaceuticals, agrochemicals, and nutrition. Even today, almost all of the world’s population depends on plantderived products. In recent times, biotechnology offers attractive opportunities for the production of plant-based in vitro systems (e.g., callus cultures, cell suspension cultures, and organ cultures) and for genetic manipulation to facilitate the generation of desired plants and plant products. As an increasing number of natural habitats are rapidly being destroyed, biotechnological in vitro techniques may help to counteract the extinction of endangered species.

Plants adapt to abiotic and biotic stresses using their astonishing plasticity to remodel themselves [1] and by the generation of secondary metabolites that are activated by elicitors and released as defense responses [2]. The generation of chemical compounds from secondary metabolism can be induced by external stress signals (e.g., pathogen elicitors, oxidative stress, wounding, etc.), which are internally mediated by jasmonate, salicylic acid, and their derivatives as signal transducers [3]. These elicitor molecules stimulate defense or stress-induced responses in plants. These can be derived from the pathogens themselves (exogenous elicitors; e.g., chitin, chitosan, and glucans) or are released by plants by the action of the pathogen (endogenous elicitors; e.g., pectin, pectic acid, cellulose, and other polysaccharides) [4]. In contrast to these biotic elicitors, there are also abiotic elicitors that act as physical agents (i.e., cold, heat, UV light, and osmotic pressure) and chemical agents (i.e., ethylene, fungicides, antibiotics, salts, and heavy metals). Elicitors modulate gene expression in response to chemical and physiologic stimuli [5]. They also induce enzyme synthesis, and thereby promote the formation of numerous secondary metabolites such as flavonoids, alkaloids, terpenoids, thionins, phenylpropanoid, and polypeptides [6].

By chance, many secondary metabolites not only reveal protective functions, but also possess medicinal value for human beings. Therefore, plant cell cultures represent interesting sources for the easy and scalable production of secondary metabolites. Approaches have been developed to optimize culture conditions and increase the yield of secondary metabolites in in vitro plant cultures. Furthermore, genetic manipulation of economic plants such as wheat, rice, maize, and others have led to stress- and disease-resistant varieties [7]. Hence, plant biotechnology may supplement traditional agriculture on an industrial scale [8].

Plant tissue culture represents an important technique in basic science and commercial application. In all major families of terrestrial plants, wounded tissue is recovered by non-differentiated callus cells. These callus cells can be cultured in vitro for biotechnological applications. Almost any part of the plant can be used to generate callus cultures. Explants taken from plant tissues slowly grow in vitro into a cell mass that ranges from amorphous and colorless to pale-brown, if they are obtained under sterile conditions avoiding microbial infection and cultured on solid gel medium supplemented with growth hormones (i.e., auxin, cytokinin). By passaging the cells regularly, callus cultures can be indefinitely maintained in vitro. Differentiated plant cells and cultured callus cells differ considerably. Callus cells are similar to non-differentiated meristemic cells; they reveal only small vacuoles and lack chloroplasts for photosynthesis, among other features. Callus cultures can re-differentiate into entire plants, if maintained under appropriate growth media that differ from standard culture media. While some callus cultures need dark growth conditions, others grow under specific day-night conditions (e.g., 16 h light, 8 h dark). Callus cultures usually grow at (25 ± 2) °C. They can be distinguished between cultures that grow in a rather compact form, and those that are friable. Friable callus cultures can be used to generate single-cell cultures that are maintained in slowly shaken liquid medium.

Plant tissue cultures can be traced back to Gottlieb Haberlandt (1854–1945), who established the first callus root or embryo cultures at the beginning of the 20th century [9]. During the 1940s and 1960s, technical advancements led to the further development of plant tissue culture techniques in order to investigate cell behavior (including cytology, nutrition, metabolism, morphogenesis, embryogenesis, and pathology), the generation of pathogenfree plants, and the conditions of germplasm storage and clonal propagation. Since the 1960s, the biosynthesis of secondary metabolites has become a subject of interest. With the advent of gene-based technological methods, novel applications were developed for callus cultures and other plant tissue technologies [10]. Plant cell cultures represent an effective means for the bioreactor-based large-scale production of therapeutically relevant secondary metabolites (e.g., anticancer drugs) [11].

The major advantages of cell culture systems, as compared with conventional whole-plant cultivation, include the following: ① The plant compounds of choice can be generated independently of external factors (e.g., soil composition or climate); ② cultured cells are not threatened by the attacks of microorganisms or insects; ③ cells of any plant—even rare or endangered ones— can easily be maintained in order to produce their secondary metabolites; and ④ robotic-driven regulation of secondary metabolite production decreases costs and improves productivity.

《2. Callus formation》

2. Callus formation

It is well known that stem cells from animal tissues usually differentiate into finally terminated tissue cells. However, it is assumed that differentiated tissue cells in plant tissues are capable of de-differentiating and regenerating wounded tissue or even the entire plant; it is also assumed that they can form totipotent callus cells [12–15]. A more recent concept claims that plant cells do not re-differentiate, but that callus is rather formed from pre-existing stem cells [16,17].

The underlying molecular modes of action that lead to stem cell differentiation and/or the differentiation–dedifferentiation of somatic plant cells are not completely understood. Stem cellrelated genes are crucial for dedifferentiation processes. Their expression is not only regulated by transcription factors, but also by epigenetic events such as histone modification and DNA methylation [18].

Arabidopsis thaliana (L.) Heynh serves as a model organism for a wide range of diverse botanical investigations. In these plants, gradients of the phytohormone auxin in embryonic callus lead to the induction of stem cell formation by regulation of the PINFORMED1 (PIN1) protein [19].

The kinetics of the spatial and temporal distribution of hormonal and developmental meristem regulators has been investigated by microscopic live-cell imaging. Relevant determinants for growth include microtubules, transcription factor networks, and cytokinin pathways, which control WUSCHEL (WUS) expression, the auxin-mediated positioning of new primordia, and so forth [20].

Numerous transcription factors control meristem formation and dedifferentiation. The transcriptional repressors WUS and WOUND INDUCED DEDIFFERENTIATION (WIND) are driving forces to maintain stem cell totipotency, while TEOSINTE BRANCHED1/ CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) is a transcriptional activator that inhibits stem cell totipotency in the shoot meristem [21].

In a time–kinetic study of immature embryo explants from maize (genotype A188), transcriptome-wide RNA-sequencing has been performed [22]. The expression of stress-related genes (glutathione-S-transferases and germin-like proteins) and hormonal transport genes (pin) was increased by more than eight-fold. Furthermore, genes related to the embryogenic growth initiation (e.g., transcription factors and receptor-like kinases) were also upregulated in a time-dependent manner. The synopsis of genes that were differentially regulated during the time course under study made it possible to build a model of the coordinated gene expression pattern in order to better understand the early steps of embryogenic culture initiation in maize [22].

《3. Culture conditions》

3. Culture conditions

Although the stem cell concept is strongly considered for callus cultures, callus cultures do not develop from isolated individual cells, but from heterogeneous structural tissues (Fig. 1). Nevertheless, callus cultures are homogeneous enough to allow micropropagation for the generation of identical copies of plants with desired features. The laboratory conditions to maintain callus cultures differ from species to species, and need to be elaborated in each individual case. An example of callus culture generation is given in Fig. 1. External factors such as light, temperature, the pH of the medium, and the aeration of cultures affect secondary metabolite biosynthesis. Usually, callus cultures are maintained on solid agar medium supplemented with specific nutrients, salts, vitamins, and elements (e.g., nitrogen, phosphorus, and potassium). In general, high ammonium ion concentrations inhibit secondary metabolite formation, while the lowering of ammonium nitrogen increases it. Inorganic phosphate is essential for photosynthesis and glycolysis. High phosphate levels often promote cell growth and primary metabolism, while low phosphate concentrations favor secondary metabolite formation. Many secondary metabolites are formed by phosphorylated intermediates, which subsequently release the phosphate; examples include phenylpropanoids and terpenoids.

《Fig. 1》

Fig. 1. Generation of callus cultures from Artemisia (A.) annua L. (a) A. annua plant; (b) A. annua leaf placed in MS98 medium; (c, d) callus growth in leaves and stipes of A. annua after 12 d, where the arrow in (c) indicates necrotic tissue; (e) callus growth in leaves and stipes after 19 d; (f) permanent callus culture; (g) callus single-cell suspension of A. annua in MS fluid medium. MS: Murashige and Skoog.

In general, the addition of precursors to the medium enhances product formation. The biosynthesis of secondary metabolites in plant cultures is usually low and needs to be enhanced in order to meet commercial purposes. The addition of precursor molecules to the medium frequently increases the product formation. The biosynthesis of most secondary metabolites consists of multistep reactions of several enzymes. Any step of the reactions in enzymatic biosynthesis chains can be stimulated to enhance product formation.

Typical culture media are well established, such as the Murashige and Skoog (MS) medium, White’s medium, and the woody plant medium [23–25]. In most cases, specific phytohormones have to be added to the medium to stimulate callus growth. To optimize secondary metabolite production, a twomedium approach is desirable: one medium for good cell growth and another for good secondary metabolite formation.

Callus formation, or somatic embryogenesis, is driven by plant hormones such as auxins, cytokinins, and gibberellins. The regeneration of whole plants from callus tissue is called organogenesis or morphogenesis. For this process, specific hormones are required as well. The similarities of hormone-starch ratios in the callus compared with the corresponding ratios in plants represent an important determinant for embryogenesis and organogenesis [26]. Growth factors such as methylglyoxal and ascorbic acid enhance insufficient organogenesis in vitro [27].

Both embryonic and post-embryonic developmental programs regulate reprograming, totipotency, and differentiation by the genetic and epigenetic mechanisms of explanted tissues and callus formation under the influence of phytohormones [28]. Callus cultures can be either embryogenic or non-embryogenic. Embryogenic callus cultures contain differentiated embryogenically competent cells that regenerate complete plants. Nonembryogenic calli contain homogenous, dedifferentiated cells, which are used for secondary metabolite production. Suspension cell cultures are frequently used for mass cultivation in specially designed bioreactors.

There are remarkable similarities between the embryogenesis of calli and the gall crown tumor formation of plants. Therefore, the molecular regulatory process in plant tumors is partially comparable to those in callus cultures. During tumorigenesis, the bacterial genome is inserted into the host genome; this activates the normal pathways of phytohormone accumulation and alters the plant’s cellular response to phytohormones. Either the phytohormones bind to their cellular receptors, leading to activated expression of downstream genes, or else T-DNAs stimulate plant cell growth, even in the absence of phytohormones [29].

《4. Production of secondary metabolites for therapeutic purposes》

4. Production of secondary metabolites for therapeutic purposes

《4.1. Bioactive phytochemicals》

4.1. Bioactive phytochemicals

A number of applications of callus cultures have commercial potential, four of which will be discussed here in more detail: ① the production of secondary metabolites for therapeutic purposes, ② the production of therapeutic antibodies and other recombinant proteins, ③ the production of agricultural plants by regeneration from calli, and ④ the production of horticultural plants by the same means.

Callus cultures may be used for the sustainable and large-scale production of secondary metabolites in pharmaceuticals, cosmetic food, and related industries. Callus cultures from medicinal plants produce bioactive phytochemicals that can be used to treat a wide variety of diseases (e.g., cancer, cardiovascular diseases, neurodegenerative diseases, infectious diseases, etc.); furthermore, the produced chemical substances do not seem to be limited to certain chemical classes, but have a wide chemical variety (Table 1) [30–78]. An example is given in Fig. 2 [67]. As phytochemicals can be directly extracted from calli without sacrificing the entire plant, the callus technology may help to protect rare and endangered plant species, and sufficient amounts of secondary metabolites can be produced in vitro. Callus cultures can also be converted to single-cell suspension cultures growing in flasks on shakers or in biofermentors in order to produce the desired secondary metabolites [79]. This allows growth under controlled conditions without the influence of varying environmental factors, seasonal variation microbial diseases, pests, and geographical constraints. Hence, secondary metabolites with constantly high quality can be produced.

《Table 1》

Table 1 Generation of phytochemicals by callus cultures derived from selected medicinal plants.

《Fig. 2》

Fig. 2. Bioactivity testing of callus cultures derived from Salvia miltiorrhiza. (a) Callus cultures from leaf and stem; (b) high-pressure liquid chromatography (HPLC) chromatograms of rosmarinic acid (RA) and salvianolic acid (Sal B) from the plant stem and stem-derived callus culture, where HPLC profiles of the plant leaves and leafderived callus cultures revealed similar results (data not shown); (c) chemical structures of rosmarinic acid and salvianolic acid; (d) cytotoxicity of steam callus extract, leaf callus extract, isolated RA, and isolated Sal B toward CCRF-CEM leukemia cells as determined by resazurin reduction assays. DMSO: dimethylsulfoxide. Reproduced from Ref. [67] with permission of Elsevier Ltd., 2016.

As callus and suspension cultures harbor the full genetic information of whole plants, they possess the totipotency for the biosynthesis of secondary metabolites. Moreover, tissue culture technologies open the possibility to manipulate the biosynthesis pathways of plant cells to produce derivatives of secondary metabolites with improved features for the market [80]. Furthermore, biotransformation reactions can be used to convert specific substrates to desired end products.

The chemical preparative techniques used to isolate and purify secondary metabolites are the same in plant cell cultures and whole plants. If the generated products are released into the culture medium, their separation is easy. If they are stored within the vacuoles, the plasma membranes and tonoplast of the plant cells have to be disrupted (e.g., by permeabilizing agents such as dimethyl sulfoxide). To optimize the production of desired phytochemicals, their biosynthesis must be up-scaled and the synthesis of unwanted side products must be minimized. Morphological differentiation and maturation favor the biosynthesis of secondary metabolites in specialized cells.

《4.2. Bioactive extracts》

4.2. Bioactive extracts

In addition to numerous reports on the production of bioactive compounds by means of callus cultures, results have been demonstrated in which extracts prepared from callus cultures perform pharmacologically interesting activities. For example, suspension cell lines have revealed growth-inhibitory activity toward colon and kidney tumor cells. Interestingly, even better results were observed from the extract than from paclitaxel and etoposide, which are established anticancer drugs [81]. The same group reported that the extract from rice callus suspension culture was also active against four cancer cell lines derived from tumors of the lung, breast, and colon. Caspase 3/7 and annexin V assays demonstrated that the extract induced apoptotic cell death. Realtime quantitative reverse transcription polymerase chain reaction (RT-PCR) of 92 genes performed with NCI-H460 lung cancer and MRC-5 normal lung cells revealed an upregulation of the CJUN, NFKB2, and ITGA2B genes in cancer cells [82].

《5. Production of therapeutic antibodies》

5. Production of therapeutic antibodies

In addition to secondary metabolites, therapeutic antibodies and other recombinant peptides and proteins have become increasingly important during the past years. Interestingly, these cannot only be produced in microorganisms (e.g., Escherichia coli, Bacillus subtilis, and Saccharomyces cerevisiae) or mammalian cell cultures (Chinese hamster ovary or baby hamster kidney cells), but also in plant callus cultures and in plant suspension cell cultures derived thereof. The latter offer advantages in terms of cost-effectiveness, large-scale production, and safety as compared with other production systems. Genes of interest (i.e., genes coding for specific therapeutic antibodies) are inserted into callus cells by Agrobacterium tumefaciens or a biolistic bombardment, also known as gene gun technology [83]. Genetically modified cells can then be used for the regeneration of whole plants under the regulation of specific phytohormone cocktails. Many proof-of-principle investigations have been published demonstrating the feasibility of this concept [84]. Some selected examples are described below.

Tobacco plants were transfected with a construct coding for an anti-phytochrome single-chain Fv (scFv) antibody. These plants accumulated high levels of scFv protein. A substantial proportion of the scFv protein was indeed functional. Transgenic callus cells secreted functional scFv protein [85].

Expression vectors harboring a scFv fragment (scFvT84.66) against the carcinoembryonic antigen (CEA) were used to transfect rice tissue. CEA is a frequently expressed biomarker for human tumors and anti-CEA antibodies are frequently used for diagnostic purposes. The amounts of antibodies were up to 14-fold higher if the antibodies were retained in the endoplasmic reticulum, as compared with the apoplast. Immunological analysis of the transgenic rice callus confirmed that the produced scFvT84.66 was indeed functional [86].

Although the 42 nm hepatitis B virus (HBV) particles are infectious, the 22 nm viral envelope surface protein is much more immunogenic. These particles have been used for vaccine production not only in yeast, but also in plants. The DNA encoding this HBV surface protein was transfected into Agrobacterium tumefaciens to generate transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.). Mice fed with transgenic lupin revealed considerable titers of HBV-specific antibodies. Human volunteers eating transgenic lettuce plants also revealed high anti-HBV surface protein IgG titers [87].

Long-term cryopreserved rice suspension cell cultures were used to generate human cytotoxic T-lymphocyte antigen 4- immunoglobulin (hCTLA4Ig). During a period of five years, hCTLA4Ig productivity in the cryopreserved cells was considerably enhanced in stably transfected cell lines [88].

Many more examples of plant-derived antibody production based on the callus culture technology could be mentioned. A previous review on this subject reported on 32 different antibodies expressed by plant biotechnology [89].

《6. Production of agricultural plants》

6. Production of agricultural plants

《6.1. Nutritional plants》

6.1. Nutritional plants

Callus culture-based technologies are not only of interest for therapeutic purposes to produce pharmaceutical secondary metabolites or therapeutic antibodies, but are also useful for agricultural and horticultural purposes. Tissue cultures reveal many genetic variations that can be used for plant-breeding programs (Table 2) [90–127]. The genetic variation to select, for example, for disease resistance is limited. Selection efficiency can be increased by in vitro selection procedures for somaclonal variants [128,129]. By in vitro selection, mutants with beneficial agronomic traits (e.g., salt or drought tolerance) can be selected in vitro and isolated for further use [130]. In addition, callus cultures play important roles in generating transgenic plants with improved features, such as resistance against draft, high temperatures, salt stress, microbial attack, and other stresses. Nutritional plants can be generated with higher yields. The examples below illustrate the potential of this technology.

《Table 2 》

Table 2 Regeneration of selected agricultural plants by callus cultures.

2,4-DA: 2,4-dichlorophenoxyacetic acid; GA: gibberellic acid; NAA: naphthalene acetic acid; BAP: benzylaminopurine; BA: 6-benzyladenine.

Salt-tolerant Shamouti orange (Citrus sinensis L. Osbeck) cells were used to regenerate plantlets. Kinetin application was required to induce plantlet formation, and root formation was induced by naphthalene acetic acid. Sodium chloride (NaCl) interfered with the regeneration process and altered the balance of phytohormones necessary for plant regeneration. The acquisition of salt tolerance was manifested in the whole plants [131].

Embryogenic calli of sugarcane plants (Saccharum spp. hybrids) were subjected to the gene gun approach using DNA-coated microparticles. Twofold gene bombardment of target tissues enhanced the transfection efficacy by more than 300. The neomycin phosphotransferase gene (npt-II) regulated by the Emu strong monocot promoter was used to select stable transformants. The calli were recovered in geneticin-containing medium, which was cytotoxic to non-transformed control calli. NPT-II protein expression in the transformants was 20- to 50-fold higher than in the non-transformed control plants [132].

Gallo-Meagher and Irvine [133] generated herbicide-resistant transgenic sugarcane plants. Gene gun-based bombardment of embryogenic calli was performed with a plasmid containing the bar gene under the control of the maize ubiquitin (Ubi-1) promoter. Shoots regenerated from transformed calli were selected for bialaphos resistance and maintained on herbicide-containing medium. Several rounds of vegetative propagation and meristem culturing resulted in herbicide-resistant clones. The transgene was stably integrated into the sugarcane genome. The mRNA expression of the herbicide-conferring bar gene was verified by RT-PCR. The transformants also exerted resistance to a commercial herbicide. This study showed that the bar gene represents a useful selectable marker for the production of herbicide-resistant sugarcane plants.

Bahgat et al. [134] described the induction of somatic embryogenesis in the calli of Vicia faba. Epicotyl- and shoot tip-derived calli were maintained in MS or Gamborg medium supplemented with sucrose, ascorbic acid, and citric acid, along with several phytohormones (benzylaminopurine (BAP), naphthalene acetic acid (NAA), and 2,4-dichlorophenoxyacetic acid (2,4-DA)). The embryos developed into plantlets, which were used for whole plant regeneration. This regeneration system will help to improve the nutritional value of faba beans.

Cassava (Manihot esculenta Grantz) is one of the largest calorie sources worldwide. Thus, cassava has been a subject of biotechnological studies to improve its nutritional content and to develop strategies for its mass production. Transgenic cassava plants have been generated as a basis for somatic embryogenesis and embryogenic callus production. To combat microbial attack, candidate genes conferring resistance to bacteria, viruses, and parasitic insects have been investigated. Future research perspectives include genome editing and novel concepts to meet the dangers posed by global climate change [135].

《6.2. Other economic plants》

6.2. Other economic plants

Biotechnological applications including callus culture technologies are also valuable for the improvement of agricultural plants not used for nutritional purposes. Two examples are tobacco (Nicotiana tabacum) and cotton (Gossypium hirsutum L.).

Early studies showed that mesophyll protoplasts of 11 different Nicotiana species could be cultured in vitro. Most of the calli originating from the protoplasts were capable of regenerating whole plants [136]. The culturing of tobacco plants as callus cultures was important in order to select for plants that exert resistance to tobacco mosaic virus infection, streptomycin, or isonicotinic acid [137–139].

Flavonoids, which protect plants from insect attack and are synthesized by the phenylpropanoid pathway, may support health in functional foods. They also have health-promotion effects when consumed by humans. Pandey et al. [140] described a technique for the cultivation of transgenic tobacco transduced with the flavonol-specific transcription factor AtMYB12 as a source of rutin production. The transgenic callus contained much more rutin than wild-type calli, resulting in significant toxicity toward Spodoptera litura and Helicoverpa armigera larvae. The authors concluded that their approach may be promising for biopesticide formulations against insect pests.

Another example is the callus-based regeneration of improved cotton plants (Gossypium hirsutum L.). Cotton calli (Gossypium hirsutum L. cv. Coker 310) have been successfully used to regenerate whole plants [141]. A significant portion of proembryoids developed from calli upon exposure to standard medium without NAA and kinetin, but with increased concentrations of KNO3 and gibberellic acid (GA) [141]. Seventeen cultivars of cotton (Gossypium hirsutum L.) were used to generate callus cultures. Embryos were derived from cytoplasmic cells and followed a predictable developmental pattern. Regenerated plants were obtained from callus cultures and were transferred to greenhouses [142].

《7. Production of horticultural plants》

7. Production of horticultural plants

Illustrative examples of the use of callus cultures in horticulture have been reported. Major crops of ornamental flowers (i.e., rose, chrysanthemum, and carnation) have been genetically modified by the transformation of embryogenic calli with Agrobacterium tumefaciens. Genes influencing shelf life, color, and resistance against disease have been transfected to them. Whole plants have also been regenerated from calli. Further flower crops used for genetic modification include Gerbera, Dendrobium, Antirrhinum, Anthurium, Eustoma, and Pelargonium [143].

Hossain et al. [144] described the selection of a salt-resistant callus line of Chrysanthemum morifolium Ramat. cv. Maghi Yellow by the stepwise increase of NaCl concentrations in the culture medium. The salt-resistant callus line revealed higher enzymatic activities of superoxide dismutase, ascorbate, and glutathione reductase activities than the salt-sensitive parental cells. A combination treatment with thidiazuron, NAA, and GA was effective for shoot organogenesis in the selected callus line. The regenerated plants did indeed reveal tolerance toward salinity stress.

Cell culture methods play an important role in rapidly multiplying the cultivars of gerbera (Gerbera jamesonii Bolus). Minerva and Kumar [145] described a protocol for indirect shoot induction from callus differentiation. In vitro plantlets were used to generate novel cultivars.

Kuehnle et al. [146] produced somatic embryos and regenerated whole plants of Anthurium andraeanum Linden ex André. Leaf explants formed calli in the dark. Embryogenesis was induced in MS medium supplemented with 2,4-DA and kinetin, as well as sucrose and glucose. Exposed to light, the calli converted into plantlets, which were transferred into pots and cultured in the greenhouse.

Petunia (P.) hybrids (P. axillaris × P. integrifolia) are economically important bedding plant crops. The floriculture industry needs protocols for marker-assisted selection breeding strategies. Guo et al. [147] described a protocol that included the use of callus cultures to identify single-nucleotide polymorphisms and cleaved amplified polymorphic sequence markers in the transcriptomes from P. axillaris, P. exserta, and P. integrifolia to characterize the genetic diversity of Petunia subspecies.

Callus cultures represent valuable tools to propagate endangered ornamental plants. Zhang et al. [148] described a protocol for somatic embryogenesis and organogenesis in Lilium pumilum under the regulation of specific phytohormones (picloram, NAA, and BA).

《8. Conclusion and perspectives》

8. Conclusion and perspectives

In the different fields of ethnobotany and ethnopharmacology, medicinal, agricultural, and ornamental plants have been used for thousands of years. The recent development of plant genetic engineering to produce plants with desirable features adds a new and growing dimension to humanity’s usage of plants (Fig. 3). It can be expected that the demand for bioengineered plants will increase in future. Many natural products are difficult to synthesize. Therefore, possibilities for the sustainable and cost-effective production of large amounts of phytochemicals are needed [149]. Many patients and customers prefer natural products to synthetic drugs. Hence, biotechnological production under controlled culture conditions in biofermentors and other culture vessels represents an attractive procedure for the commercial mass production of phytochemicals and therapeutic antibodies, as well as regenerated nutritional plants from calli. More and more knowledge is being gathered about the biosynthetic pathways of desirable natural products, which will further enhance the development of bioengineering and gene-based technological engineering techniques for callus cultures from medicinal plants, including plants used in Chinese medicine. The same is true for the generation of genetically modified plants in agriculture and horticulture. Although the full potential of callus plant culture technology has not yet been exploited, the time has come to develop and market more callus culture-based products.

《Fig. 3》

Fig. 3. A synopsis of biotechnological applications for callus cultures.



The author would like to personally thank Mrs. Doris Rohr for her excellent technical assistance and commitment during the past decade.