Advances in Plant Tissue Culture: Current Developments and Future Trends

By Ajay Kumar and Arpan Modi

Advances in Plant Tissue Culture: Current Developments and Future Trends provides a complete and up-to-date text on all basic and applied aspects of plant tissue cultures and their latest application implications. It will be beneficial for students and early-career researchers of plant sciences and plant/agricultural biotechnology. Plant tissue culture has emerged as a sustainable way to meet the requirements of fresh produces, horticultural crops, medicinal or ornamental plants. Nowadays, plant tissue culture is an emerging filed applied in various aspects, including sustainable agriculture, plant breeding, horticulture and forestry.

This book covers the latest technology, broadly applied for crop improvement, clonal propagation, Somatic hybridization Embryo rescue, Germplasm conservation, genetic conservation, or for the preservation of endangered species. However, these technologies also play a vital role in breaking seed dormancy over conventional methods of conservation.

• Focuses on plant tissue culture as an emerging field applied in various aspects, including sustainable agriculture, plant breeding, horticulture and forestry
• Includes current studies and innovations in biotechnology
• Covers commercialization and current perspectives in the field of plant tissue culture techniques

• Language: English
• Publisher: Academic Press
• Release date: May 28, 2022
• ISBN: 9780323998192

Book preview

Chapter 1: A general introduction to and background of plant tissue culture: Past, current, and future aspects

Md Intesaful Haquea; Prashant Kumar Singhb; Sandip Ghugec; Anil Kumard; Avinash Chandra Raic; Ajay Kumare; Arpan Modic,⁎    a Fruit Tree Science Department, Newe Ya’ar Research Center, Agriculture Research Organization, Volcani Center, Ramat Yishay, Israel

b Department of Biotechnology, Mizoram University (A Central University), Pachhunga University College Campus, Aizawl, Mizoram, India

c Institute of Plant Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel

d Department of Nematology, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel

e Post-harvest and Food Sciences, Agricultural Research Organization, Volcani Center, Rishon LeZion, Israel

⁎ Corresponding author. e-mail address [e-mail]

Abstract

Plant tissue culture comprises all the ways to nurture the plant cell, tissue, or organ in a specific manner under an artificially created environment to produce uniform planting material in bulk. It also covers the ways by which one can improve the traits of the plant. Plant production outside the laboratory environment can be achieved through seeds, utilizing zygotic cells, or cuttings, through somatic cells. Thus, it is noteworthy that somatic cells have at least the potential for regeneration. In the case of cuttings, the tissue involved in the propagation procedure is meristematic tissue. However, under in vitro conditions, both the zygotic and somatic cells (including nonmeristematic tissues) contribute towards plant propagation. In vitro conditions or artificial environments are the key difference between plant propagation in field and plant propagation in laboratory. Such propagation under a controlled environment (micropropagation) leads to the mass multiplication of plants. The present chapter introduces the technique and emphasizes several factors controlling plant growth under in vitro conditions. The chapter also covers how plant tissue culture techniques can be utilized for plant production and trait improvement.

Keywords

Artificial environment; Mass multiplication; Micropropagation; Plant tissue culture; Soil-less cultures

1: Introduction

Plant tissue culture is a promising tool for plant production and trait improvement, and has secured a significant position in biotechnology. It is based on the principle of totipotency, which is defined as the ability of any plant cell to convert into a whole plant if appropriate conditions of nourishment are provided. This definition includes somatic cells also, which undergo a series of developmental changes leading to organogenesis. All the developmental processes can be seen in small culture vessel and the provided environment is said to be in vitro conditions. This environment comprises a medium on which plant cells, tissue, or organs are nurtured, and an aseptic condition to ensure contamination-free culture. Being a eukaryotic organism, a plant has distinct parts with distinct morphological, biochemical, and physiological features. Correspondingly, all the parts are made up of different functional units which are genetically similar but function differentially (Hussain et al., 2012). Due to their distinct functional existence, they also respond differentially to tissue culture conditions. Some tissues like meristems are highly active and used widely for commercial propagation.

In contrast, some tissues present in the leaf and internodes are used for the regeneration purposes required for the transformation studies. A schematic representation of the tissue culture process is shown in Fig. 1. The process starts with establishing the culture through seed, root leaf, flower, internodes, or meristematic tissues. Since the plant part (known as explant) is taken from the outside environment, it should be made free from microorganisms to establish the axenic culture. For this purpose, various sterilizing agents like bactericides, fungicides, ethanol, sodium hypochlorite, and mercuric chloride are used. After the surface sterilization procedure, the culture established from the root, leaf, flower, and internodes may be subjected to direct or indirect (callus-mediated) organogenesis. Seeds may be grown until the seedling stage from which cotyledonary leaf or hypocotyl (representative of internode) may be taken for organogenesis or from the developed plantlet; meristematic tissue can be taken. Direct or indirect organogenesis leads to the formation of shoots which are further proliferated to form other plant parts (not reproductive). Fully developed plantlets are then taken out for hardening and acclimatization. The explant is exposed to essential macro- and micronutrients, vitamins, and plant growth regulators throughout the micropropagation process. If we look at the developmental process of propagation of explant, the cells undergo continuous formation of cell division through mitotic cycles. Although genetic factors control such divisions, some epigenetic changes may occur during this period.

Fig. 1

Fig. 1 Schematic representation of tissue culture process through different organs.

Moreover, using some plant growth regulators may also lead to mutation; due to such changes, genotypic and/or phenotypic variations are induced in the generated plantlets. These variations are called somaclonal variations—monitoring of such variations during micropropagation or before the plants become ready for selling. Molecular markers like DNA, RNA, and protein (or sometimes metabolite-based) detect polymorphism (variations) among individuals in the population. In this case, the variations that have developed between the mother plant and micropropagation plants due to tissue culture manipulations are checked with the help of DNA-based markers (Alizadeh et al., 2015).

The scope of plant tissue culture is vast. Fig. 2 shows all the possible products for which plant tissue culture techniques may be used. Briefly, the technique has two fates: production and improvement. Production comprises healthy, competent, superior-quality uniform planting material, production of synthetic seeds, and secondary metabolites for pharmaceutical applications. On the other hand, improvement can be made through transformation, haploid plant production, enhancing ploidy level, protoplast isolation (for transient expression of a gene of interest), and fusion (for interspecific hybrids). Apart from these two significant scopes, plant tissue culture is also a good platform for cryopreservation, somaclonal variations, and breaking seed dormancy. The procedure (Fig. 1) in the background shows that distinct aspects of plant tissue culture are discussed here. Prerequisite parameters essential for establishing tissue culture of any plant species are selection of superior-quality, disease-resistant plant and explant type according to the need. For commercial multiplication of plant, shoot tips or nodal segments from juvenile phase are used; for direct or indirect organogenesis or somatic embryogenesis, young leaves (especially in vitro leaves) are used. In vivo explants other than seed, shoot tip, or nodal segments are avoided for the culture establishment as the tissue becomes highly differentiated, and forcing them into de-differentiation and re-differentiation may impart physiological stress to the explant. Apart from the leaf tissue (in vivo), release of secondary metabolites in the medium and subsequent detoxification is also a major problem.

Fig. 2

Fig. 2 Scope of plant tissue culture from production and improvement perspectives.

Moreover, selection of genotype is especially important. For any plant species, many cultivars can be found. Selection of superior planting material can be made according to the product of interest. For instance, medicinal plants are used for secondary metabolites. Thus, selection criteria should also include the concentration and type of the metabolite of interest apart from the general disease resistance and vegetation vigor (if the important plant part is a leaf). After selecting a suitable explant, the next important operation is to make it free of microbes and all types of contaminants. Contaminants can be dust particles, bacteria, fungi, and other microbes. For this process, 70% alcohol, bactericides, fungicides, and other chlorides such as sodium hypochlorite and mercuric chloride may be used. Because such chemicals can also harm the plant tissues if used for a prolonged time, their concentrations and time should be optimized before commercial application (Bhatia et al., 2015a, b). Nourishment of the aseptic plant parts begins after successful elimination of all the contaminants. This is facilitated by culture environment. Broadly, the culture environment can be divided into physical environment and chemical environment. Physical environment includes temperature, light, air passage, physical boundaries of culture vessels, and physical properties of the culture environment, which include pH of the medium; humidity inside and outside the culture vessel; fluidity of the culture medium; temperature of the growth room; light types; and duration (photoperiod) and aeration. The effect of different physical factors on growth and performance of other plants under in vitro conditions are mentioned in Table 1. The requirement of medium pH for different plant species may vary, but the ideal pH range in which most of the plant can be grown is 5.5–6.5. A medium with lower pH may be subjected to loosening of texture and facilitates less uptake of nitrates for plant growth.

Table 1

In contrast, higher pH may make the media very hard, and the availability of the medium components will be affected. Experiments comprising relative humidity are difficult to execute as the explant tends to contribute to humidity as it grows in the culture vessel. The presence and absence of gelling agents also contribute significantly towards plant growth. The lack of the gelling agent is advantageous in terms of aeration, nutrient availability, and cost reduction, whereas contamination and vitrification are major disadvantages of it. Most plant species respond well in 22°C–25°C. Some exceptional cases may be seen where increasing temperature increases the plant’s growth (Kaur and Mudgal, 2021). Lowering the temperature along with changing media composition is beneficial for the long-term storage of the plant material. The last and most important physical factor is light. Light contains spectra of assorted colors. Spectral distribution of all the color in white fluorescent varies, whereas in LEDs it is specific. Apart from light, the duration of light and dark period is considered in many studies. According to the plant’s photoperiod, different light and dark hours, viz., 12 h/12 h, 14 h/10 h, 16 h/8 h, and 24 h light or dark, may be set for the experiment.

On the other hand, chemical factors of culture environment include macro- and micronutrients, vitamins, carbon source, plant growth regulator (PGR), gelling agent, and growth modulator other than PGR. Plant tissue culturists focus on these aspects of the culture conditions the most during optimization of the protocol. Starting from the application of different predefined media such as MS, LS, WPM, and B5, to different concentrations of various gelling agents such as agar, bacto-agar, phytagel, and gelrite are taken for the consideration in research as well as commercial optimizations. Apart from these, some antioxidants and/or adsorbents such as phenolic acids, ascorbic acid, PVP, and charcoal are also used in the medium according to the protocol’s requirement. Medium composition has many possibilities, both quantitatively and qualitatively. One may find a complete book on this single factor (Saad, 1988; Herman, 2008), or a particular medium component such as meta-topolin (Ahmad and Strnad, 2021), TDZ (Ahmad and Faisal, 2018), etc. The requirements of macro- and microelements, plant growth regulators, carbon source, and gelling agent vary from plant to plant. Even after optimization of nutrient composition for particular plant species, there is always scope for improvement. This can be seen from various research papers on the same plant species where we can find different composition in different articles. Improvement of the protocol not only includes changing the nutrient composition of the medium, but also may be done through the way organogenesis is achieved. In this book, two chapters (Chapters 3 and 4) are focused on this aspect. In general, MS, B5, and WPM are some of the commonly used basal media; BA, kinetin, TDZ, IBA, IAA, 2,4-D, and GA3 are commonly used plant growth regulators alone or in combination with different concentrations, whereas sucrose and agar are the carbon source and gelling agent, respectively, used most.

2: Milestones

The idea of totipotency of the plant cell was recognized a century ago. A German botanist, Gottileb Haberlandt, in 1902 proposed the principle of totipotency and has been called the father of plant tissue culture (Haberlandt, 1902). However, his experiment on Tradescantia cell culture failed as the cells could not undergo cell division. There were a few observations (before the 19th century) that can be considered as initiators of such a concept. Henri Louis Duhamel du Monceau observed in 1756 that elm plants, when wounded, showed healing by forming an unorganized mass of cells (now called callus). In 1878, Vöchting observed that the upper part of the shoot produces buds and the basal part produces callus or roots; he suggested the polarity of growth substances (now called polar distribution of auxin) as a key factor in plant growth and development. Many authors have reviewed the history of plant tissue culture techniques with the theme of significant achievements of past researchers and current commercialization (Thorpe, 2007, 2012; Aladele et al., 2012; Sussex, 2008; Twaij et al., 2020). In the last two decades, after successful trials of biotransformation in model and nonmodel plants, the technique of plant tissue culture has become an inevitable part of plant biotechnology as most of the cases of transformation may be seen as tissue culture dependent. Milestones achieved within the 20th century are described in Table 2.

Table 2

3: Direct and indirect organogenesis

The term Organogenesis is defined as the formation and the development of the organs of an organism from embryonic cells on a suitable medium. In plant tissue culture, the process of organogenesis has provided useful systems to learn about the regulatory mechanisms of the plant development (Hicks, 1994; Bhatia and Bera, 2015). It involves using plant materials such as root leaves, stems, and cotyledons as explants to induce organogenesis with specific growth mediums and combinations of plant growth regulators (PGRs) or phytohormones (Parab et al., 2021).

Organogenesis can be split into three stages (Dodds and Roberts, 1985). In the first stage, explants obtain competence, which is defined as the ability to respond to hormonal signals of organ (shoot bud) induction. During this stage, the process leading to organogenic competence is called dedifferentiation, where differentiated cells develop into an undifferentiated mass. In the second stage, competent cells in cultured explants are destined and determined for specific organ formation under the influence of the combinations of PGRs or phytohormones. Finally, the third stage is where morphogenesis proceeds independently of the exogenously supplied phytohormones. Thus, plant organs take the shape of roots or shoots when phytohormones are removed from the culture medium (Miesfeld and Brown, 2019).

The propagation methods of tissue culture are direct organogenesis with no intervening callus stage, which can be achieved from shoots that directly originate from the meristematic regions of explants. Indirect organogenesis and different callus types obtained from explants were subcultures on the MS medium with varying cytokinin concentrations for shoot and auxin for rooting. The callus phase embryo that arises at the preliminary stage is not considered an organ because of the absence of a vascular system and its independent existence (Huang et al., 2020). Several explants, including cotyledonary nodes (Arun et al., 2014), leaf (Arshad et al., 2012; Yang et al., 2006; Pourhosseini et al., 2013), mature internodal stem segments (Curtis and Mirkov, 2012; Rani and Dantu, 2012), apical shoot buds (Rathore et al., 2020), and stems with nodal segments (Rani and Dantu, 2012; Pérez-Jiménez et al., 2012) have been reported for the development of direct and indirect organogenesis. Direct organogenesis in many plants is a limitation in callus subculture resulting in genetic diversity and undesirable variation of clones known as somaclonal variation (Takagi et al., 2011). The sterilized explants segments were inoculated on an MS medium supplemented with different concentrations of cytokinin (thidiazuron or TDZ, 1.0, 2.0, 3.0, 4.0, 5.0 mg L−   1) for direct shoot regeneration for 4 weeks. Direct organogenesis is commercially important for production and regeneration, omitting the callus induction phase, which was particularly desirable in modern breeding where it was necessary to grow rapidly and reduce the regeneration cost (Pourhosseini et al., 2013).

Indirect organogenesis is the plant developmental process in which plant cells develop from a callus mass (tissue formation on an explant cut/wounded site). The different callus types achieved from explants were subcultured on a fresh MS medium with different concentrations of cytokinin BAP (1.0, 2.0, 3.0 mg L−   1) for shoot regeneration. Indirect organogenesis is commonly used to develop transgenic plants from transformed callus/explants. The initial explant is transformed, and the callus and shoots are developed from the modified explants. Typically, indirect organogenesis is more critical for transgenic plant production (Singh et al., 2016; Kumari et al., 2017). In some cases, a medium supplemented with a combination of cytokinin 6-benzylaminopurine (BAP) and auxin α-naphthalene acetic acid (NAA) has been suggested for callus induction and subsequent formation of adventitious bud (Scaramuzzi et al., 1999; Zhou et al., 2000).

4: Somatic embryogenesis

Somatic embryogenesis is a developmental process where plants can regenerate bipolar structures from a somatic cell (Von Arnold et al., 2002; Méndez-Hernández et al., 2019). During this process, plant somatic cells (explants) can dedifferentiate to a totipotent embryonic stem cell, giving rise to an embryo under aseptic and control conditions. Thus, the newly developed embryo can further produce into a whole plant (Guan et al., 2016).

Plants exhibit remarkable regeneration capacity, ensuring developmental plasticity; they regularly need to regenerate in response to physical damage caused by their biotic or abiotic environment (Fehér, 2019). Plant somatic cells can be reprogrammed to form a pluripotent cell mass called the callus. Totipotent has two different clarifications: (a) capable of developing into a whole organism or (b) capable of differentiating into any cell types of an organism (Condic, 2014). A portion of pluripotent cells (callus) gives abundant shoots via de novo shoot organogenesis (Shin et al., 2020).

To understand the physiological and molecular mechanisms for the induction of somatic embryogenesis (direct or indirect) is necessary for its manipulation (Grzyb et al., 2018). Multiple factors can influence somatic embryogenesis. Conditions of culture medium, the high amount of plant growth regulators (PGRs), and wounding to the explant are other types of stress that can cause plant tissue to change its cellular and molecular mechanism. The explant age and its type can influence the success of somatic embryogenesis. In particular, the yield of young explants has more somatic embryos than older explants. The embryogenic calli arise at different frequencies from different explant tissues (roots, flowers, shoots, etc.) from the genotype of the mother plant. Furthermore, explants coming from different cultivars/varieties of the same species may present different responses to somatic embryogenesis induction (Pěnčík et al., 2015; Loyola-Vargas and Ochoa-Alejo, 2016).

PGRs supplement in culture media plays a vital role in inducing cell differentiation, individually during the induction of somatic embryogenesis (Méndez-Hernández et al., 2019). Auxins help promote callus proliferation and inhibit differentiation. Removing or decreasing the concentration of auxin will help in the development of somatic embryos. Auxins are also responsible for establishing cell polarity in the embryo (apical and basal axis). Auxins supplement in culture media either alone or combined with other PGRs, which induces the expression of different genes, which modify the genetic program of the somatic cells and regulate the transition to each of the stages during the development of somatic embryogenesis (Loyola-Vargas and Ochoa-Alejo, 2016; Awasthi et al., 2017; Márquez-López et al., 2018). However, cytokinins are also suitable candidates for the induction of somatic embryogenesis. In general, it has been suggested that cytokinin, like thidiazuron (TDZ), is more effective than other cytokinins for somatic embryogenesis (Ioja-Boldura et al., 2010Reyes-Diaz et al., 2017; de Almeida et al., 2012; Ahmed and Anis, 2012). In some plants, minor components such as amino acids (glutamine, proline, tryptophan), polyamines (putrescine, spermidine), and brassinosteroids have been reported to enhance somatic embryogenesis in some species (Rathore et al., 2020; Akin et al., 2018; El-Dawayati et al., 2018; de Moura et al., 2019; Jin et al., 2020; Raju et al., 2020; Peng et al., 2020).

Light conditions play a significant role when working with somatic embryogenesis. Initial explant cultures need to be maintained in darkness at 25°C for 6 weeks (Rathore et al., 2020; Corredoira et al., 2002). White light develops growth, but at the same time produces a prominent level of phenolic compounds and abscisic acid in the medium. These materials induce oxidative reactions and as a result the tissue becomes brown (Pinto et al., 2008). To avoid these light-derived effects, activated charcoal is used in the medium (Rathore et al., 2020; Lelu-Walter and Pâques, 2009). Under this scenario, light conditions should be tested for somatic embryogenesis for the maturation stage.

Maturation is a preliminary stage for embryo development, which is obligatory for significant germination. It is one of the crucial stages of embryogenesis. During maturation, water loss is a major factor in tissue culture treatments. It promotes the maturation of the embryo using osmoticums like sucrose (Wang et al., 2019b). Sucrose is a source of energy and reduces the water potential, which eventually leads to water deficit of the culture medium (González et al., 1995; Javed and Ikram, 2008). Numerous studies can be found for both organogenesis and somatic embryogenesis. However, pros and cons are always associated with both routes (Table 3).

Table 3

Somatic embryogenesis has four phases:

•The first stage is where explants (callus retaining cells with embryogenic competence) initiate development of vegetative cells or tissues.

•In the second stage, a group of cells with an embryogenic fate, i.e., embryogenic cell lines, are maintained and developed.

•The third stage involves the formation of somatic embryo (embryo undergoes globular, heart-shaped, torpedo, and cotyledonary stages) and maturation (accumulation of reserve substances).

•Finally, somatic embryos are converted (germinated) into viable plantlets.

5: Plant tissue culture for trait improvement

Plant tissue culture is a powerful tool for developing industrial important new plant traits and varieties and producing on a large scale and high yield to achieve the demand, often to produce the clones of plants (Naik et al., 2020). Plant tissue culture techniques are a leading supportive tool for plant breeding and genetic engineering programs. Plant tissue culture aims to improve desirable characteristics in plants using various techniques, such as anther/microspore culture, somaclonal and gametoclonal variation (Ziauddin and Kasha, 1990; Bhatia et al., 2015a, b), embryo culture via haploid production (Morrison et al., 1991), protoplast culture (Loyola-Vargas and Ochoa-Alejo, 2018), somatic hybridization (Pratap et al., 2010), and transgenic plant development (Singh et al., 2016; Kumari et al., 2017), which are being used to generate genetic variability towards crop improvement. Modern agriculture demonstrates exactly how plant science research and technology can come together to improve crop yield and quality. However, nowadays, the conventional breeding technique is much faster than it was 50 years ago (Gao, 2018).

In recent years, plant tissue culture techniques have gained major economic importance in plant multiplication, disease elimination, insect resistance, and plant improvement (Hussain et al., 2012; Asaf, 2019; Chadipiralla et al., 2020). Plant tissue culture technology is being used widely for large-scale plant propagation. Small pieces of tissue can be used to produce hundreds and thousands of plants in a continuous process. A single explant can be divided into several million plants for the ornamental market in a relatively short time period under controlled conditions, throughout the year and regardless of weather and climate dependence (Chugh et al., 2009). The conservation of rare and endangered species has been carried out successfully by micropropagation because of the high coefficient of multiplication and small requirement of the starting material and space (Oseni et al., 2018). In combination with molecular techniques, tissue culture techniques have successfully incorporated specific traits through gene transfer (Patel et al., 2015; Singh et al., 2016; Kumari et al., 2017). Secondary metabolites production, like food colors, dyes, food flavors, fragrance, drugs, natural products, and scented oils used in aromatherapy through cell cultures/hairy root cultures, are leading examples of molecular farming/pharming (Gosal and Kang, 2012; Bhatia et al., 2015a, b).

Anther culture is an in vitro technique for rapidly developing fully homozygous inbred lines haploid embryos called embryoids, aseptically under artificial nutrient medium (Mayakaduwa and Silva, 2018; Tripathy et al., 2019). Anther culture avoids several inbreeding cycles and the lengthy process used in traditional selfing methods, including bud pollination (Ali et al., 2021). Phenotypes in haploid plants by anther culture are easily recognized by their small sterile flowers (Filiault et al., 2017). Anther culture was first reported in the 1970s through in vitro methods (Guha and Maheshwari, 1966). It is increasingly being used to improve cereal crops, both as a source of haploids and to induce new genetic variation (Hassan and Islam, 2021). It has been used in various species, and also to improve certain vegetable crops such as asparagus, sweet pepper, tomato, eggplant, watermelon, chickpea, and Brassica vegetables, but mainly Oryza sativa (rice) and Nicotiana tabacum (tobacco) (Cao et al., 1994; Emrani Dehkehan et al., 2017; Kurtar, 2017; Heidari et al., 2017; Reed, 2018; Niazian et al., 2019; Tripathy et al., 2019; Sood et al., 2021; Abdollahi and Seguí-Simarro, 2021; Alan et al., 2021; Sari and Solmaz, 2021; Palacios and Seguí-Simarro, 2021; Ali et al., 2021). Advantages of anther culture include a high frequency of haploid plants; cell division can be easily induced in most species. Haploid production (embryoids) or plantlets offer a better line with high yield resistance to disease. It is an essential tool for physiological and genetic researchers in their agriculture fields (Tripathy et al., 2019; Kharate et al., 2021). Due to only one set of chromosomes, even the recessive mutations are immediately expressed in haploids. In several crops, desirable mutants have been isolated among haploids derived in culture. A high level of expertise is not needed.

Somatic cell hybridization is one of the most important tools of protoplast culture (Fehér and Dudits, 1994). Protoplasts are living plant cells without cell walls (naked cells); cell walls are removed by physical means or with specific combination of lytic enzymes (Bilkey and Cocking, 1982). This is specifically significant for hybridization between genetically distinct species/genera, which cannot be made to cross through traditional methods of sexual hybridization, and to bring about genetic recombination and develop hybrid genotypes (Kästner et al., 2017; Anné and Peberdy, 2020; Raman et al., 2021; Kim et al., 2021; Bruznican et al., 2021). The protoplast culture technique is one of the most frequently used methods in plant tissue culture research, by molecular biologists, biochemical engineers, and biotechnologists (Mastuti and Rosyidah, 2018; Zeng et al., 2021; Ahmed et al., 2021). Somatic hybridization aims to transfer desired traits that produce beneficial novel crop plants without genetic transformation/engineering, such as nitrogen fixation (Saha et al., 2017), drought resistance (Ishaku et al., 2020), fast growth and high yield production rate (Evans, 1983; Gurel et al., 2002), protein quality (Xie et al., 2020), and heat and cold resistance (Verma et al., 2008). Production of fertile diploids and polyploids from sexually sterile haploids, triploids, and aneuploids is crucial in advancing crop improvement. Transfer genes for disease resistance (Omar et al., 2017), abiotic stress resistance, herbicide resistance (Li et al., 2020), agronomically important genes (Zhang and Wu, 1988), and many other quality characteristics through the integration of protoplast are increasing crop improvement (Begna, 2020).

Embryo rescue is a highly efficient technique used for the development of a plant from an immature embryo that does not survive properly to develop into a complete plant, which means curing inherently weak, immature, starved, or hybrid embryos to prevent degeneration before an abortion (Sharma et al., 1996; Olivares-Fuster et al., 2002). In the early nineteenth century, a successful culturing method of plant embryos under aseptic conditions was performed (Hannig, 1904). Embryo rescue is economically important, reducing the cost of developing desired interspecific hybrids through embryo rescue in Brassica species (Ripa et al., 2020). Embryo rescue can efficiently produce interspecific hybrids and the development of Brassica vegetables (such as cabbage) with new traits (Pen et al., 2018). To date, the embryo rescue method has been applied in grape breeding for more than three decades (Li et al., 2015). The most common application for embryo rescue in grape breeding is to cure inherently weak embryos, and early maturing and muscadine grapes, exhibiting very poor germination potential due to seed nutritional and/or physiological deficiencies (Li et al., 2014; Emershad and Ramming, 1984; Guoyin-Shan et al., 2004; Ledbetter and Shonnard, 1990; Ponce et al., 2002; Ramming et al., 1991; Tsolova and Atanassov, 1994), Recently, fruit breeding programs have greatly increased the interest in developing improved seedless fruits (Emershad et al., 1989; Burger and Goussard, 1996; Nookaraju et al., 2007).

Agrobacterium-mediated (A. tumefaciens) gene transfer is one of the most efficient methods of gene transformation in plants to date. It has been widely used to develop transgenic plants to study the function of genes improving yield agricultural traits, for example, resistance to diseases and insects, tolerance of abiotic stress (drought and salt), and higher quality and yield (Singh and Kumar, 2021; Singh et al., 2016; Kumari et al., 2017). In general, Agrobacterium-mediated transformation is a tissue culture-based method, which usually requires an aseptic condition and regenerates shoots and roots from the transformed tissues to develop a complete plant (Jagdish and Koundal, 2020; Jha et al., 2021). The tissue culture technique and Agrobacterium-mediated gene transfer contribute towards the modern agricultural science for the study of functional genes that have been used in model plants such as Arabidopsis and Nicotiana (Kumari and Jha, 2019; Tiwari and Bae, 2020; Dubey et al., 2021; Singh and Kumar, 2021). The naturally occurring A. tumefaciens plays a critical role in gall induction via Ti plasmid (tumor inducing size >   200 kb) by infecting wound sites in dicot plants, leading to pathogenic development of crown galls (Hoekema et al., 1983).

Genetic engineering is a highly gold stander efficient technique used for development with specific characteristics (trait) to protect against disease, biotic and abiotic stress, and cold and heat, and it increases crop yield in transgenic plants. Salinity and drought stress are important environmental factors that reduce crop yield. Much research led to the development of transgenic plants that incorporate salt- and drought-resistant genes that reduced the effects of salinity and drought stress in several transgenic plants (Patel et al., 2015; Pandey et al., 2015; Tiwari et al., 2016; Pandey et al., 2016; Singh et al., 2016; Kumari et al., 2017; Rezaei Qusheh Bolagh et al., 2021). Development of transgenic virus resistance has been achieved over the past decade, in various crop virus-resistant plants developed to protect from significant threats of viral disease in crops around the world (Prins, 2003; Yang et al., 2019; Zhan et al., 2019; Zhao et al., 2020; Kalinina et al., 2020). Transgenic technology reduced the applications of toxic insecticides and herbicides throughout millions of hectares worldwide (Manickavasagam et al., 2004; Wang et al., 2017; Javied et al., 2021; Li et al., 2021). Moreover, a cold stress resistant/tolerant plants have also been developed using transgenic technology (Luo et al., 2017; Su et al., 2017). Additionally, genetic transformation is also a useful tool to develop a transgenic line that introduced C4 pathway genes to improve photosynthesis, facing global warming and rising CO2 levels in the atmosphere (Gao et al., 2018; Ermakova et al., 2021; Swain et al., 2021). Production of plant secondary chemicals through metabolic engineering (Pouvreau et al., 2018) and the regulation of the plant metabolic pathways to get the desired products have become promising tool for the pharmaceutical industries (Sartaj Sohrab et al., 2017; Loyola-Vargas and Ochoa-Alejo, 2018; Espinosa-Leal et al., 2018).

Promising uses of the CRISPR/Cas9 genome editing tool one of the technologies in agriculture involve revolutionizing the gene knockout/breeding and improvement programs of several crops. Over the past decade, several genome editing techniques combined with plant tissue culture based on the use of sequence-specific nucleases have allowed precise manipulation of target gene sequences to create specific desirable mutations (Gao, 2018; Sedeek et al., 2019; Kalinina et al., 2020; Zhang et al., 2021). Moreover, many crop species are resistant to regeneration through tissue culture (Eş et al., 2019).

6: Plant tissue culture for plant production

Plant production is aimed towards commercial production of planting material and its products, such as secondary metabolites. This section discusses three aspects of plant production, viz., planting material, secondary metabolites, and synthetic seeds. In a commercial tissue culture industry, plant production is conducted in a plant with a meager germination rate, or conventional methods of propagation are not effective for plant production. Plant-like date palm, papaya, and some vegetables are also targeted as they are dioecious and the fruit will only be set on female flowers. Therefore, tissue culture-raised female plants can almost double the production of a seed-raised population (where 50% of the plants are expected to be males). However, males are required for pollination. Dioecious plants have male and female flowers separately, and a fruit, produced only on females, is the producers’ ultimate goal. However, a seed-raised population will not indicate anything about the gender of the plant. Expecting half of the population to be female, seedling-based farming renders half of the land unproductive. Vegetative propagation from the female plants may be carried out, but it has limitations with the planting material. In such cases, tissue culture technology is helpful. Plant dioecy can be seen in diverse groups of plants such as vegetable, horticultural, and medicinal plants. Some tissue culture protocols for plants with, lower germination rates; lower production through vegetative means; and dioecious nature are mentioned in Table 4.

Table 4

As a sessile organism, the plant produces a variety of chemicals known as secondary metabolites to survive in adverse environmental conditions. These conditions can be biotic or abiotic or both. These metabolites are often useful for different industries such as agricultural, pharmaceuticals, textiles, and food and beverages. They are found in different organs of the plant, and in some cases, all the parts of the plant. Plants are a reliable source of such chemicals compared to their synthetic counterparts. In some cases, these chemicals serve as raw materials for synthetic compounds (Karuppusamy, 2009). Secondary metabolites are produced exclusively in plants as they contain dedicated biosynthetic pathways. They differ from the primary metabolites because they do not take part directly in the growth and developmental processes (Croteau et al., 2000). Often, these compounds are produced from the end or intermediate products of the primary metabolism. Like primary metabolites such as carbohydrates, protein, and lipids, secondary metabolites are also made up of C, H, O, and N. However, many do not contain nitrogen atoms. Therefore, they can be classified as nitrogen-containing (alkaloids, nonprotein amino acids, amines, cyanogenic glycosides, glucosinolates) or without nitrogen (terpenoids, phenolics, flavonoids, polyketides). Production of such compounds through farming has a few disadvantages like land scarcity, slow growth, environmental influence, and disease infestations. These factors affect both production and productivity (Thiruvengadam et al., 2016). Alternative to field production, these chemicals can be produced in vitro, which overcomes all the disadvantages associated with conventional means of production (Espinosa-Leal et al., 2018). Various alkaloids, terpenoids, steroids, quinones, and phenylpropanoids are produced (Chandran et al., 2020). Secondary metabolites can be produced by three different routes: organ culture, callus culture, or cell suspension culture. Organ culture includes hairy root culture and shoot culture. When explants are infected with Rhizobium rhizogenes (previously known as Agrobacterium rhizogenes), hairy roots obtrude from the site of infection. The resulting structure is massive and in the state from which important metabolites can be extracted. Due to this, commercial production of industrially important metabolites is feasible. Often the production is enhanced through the application of elicitor molecules. They are signaling molecules for which plant cells contain receptors. After recognizing this receptor, a defense mechanism is fueled by the overproduction of secondary metabolites (Tien, 2020). Some of the examples include production (through adventitious root culture) of ginsenoside from Panax ginseng (Marsik et al., 2014), glycyrrhizic acid from Glycyrrhiza uralensis(Wang et al., 2019a), saponin from Panax vietnamensis (Linh et al., 2019), anthraquinone, phenolics, and flavonoids from Morinda citrifolia (Baque et al., 2012), hyoscyamine and scopolamine from Datura metel (Ajungla et al., 2009), capsidiol from Capsicum annum (Chávez-Moctezuma and Lozoya-Gloria, 1996), L-DOPA from Hybanthus enneaspermus (Sathish et al., 2020), and hypericin from Hypericum perforatum (Tavakoli et al., 2020). The production of secondary metabolites through hairy root cultures includes azadirachtin from Azadirachta indica (Satdive et al., 2007), plumbagin from Plumbago indica (Gangopadhyay et al., 2011), glycyrrhizin from Glycyrrhizia inflata (Putalun et al., 2011), artemisinin from Artemisia annua (Ahlawat et al., 2014), ajmaline from Rauwolfia serpentina (Srivastava et al., 2016), and ursolic acid and eugenol from Ocimum tenuiflorum (Sharan et al., 2019). Another approach to producing high-valued secondary metabolites is shoot culture. Just like the aim of hairy root culture to maximize the root biomass, shoot cultures are expected to give a higher multiplication rate within a short time frame (Bourgaud et al., 2001). Several factors need to be considered and optimized to maximize production. These include: a selection of cell lines; medium composition and choice of elicitors, inoculum density; and physical factors of culture environment (Murthy et al., 2014). Production of cardenolides (Pérez-Alonso et al., 2018), flavonoids (Kawka et al., 2021), hyperforin (Coste et al., 2021), dibenzo cyclooctadiene lignans (Szopa et al., 2021), and phenolic acids (Ekiert et al., 2021) are some examples of organ cultures. Compared to organ cultures, callus or cell suspension cultures do not require differentiated cells or tissue. They are an unorganized mass of cells and are mostly carried out in liquid culture systems such as bioreactors (Bourgaud et al., 2001). The use of callus cultures for secondary metabolites production and several other biotechnological applications is discussed in detail by Efferth (2019). Cell suspension culture can be produced from callus culture and has very good potential for the biosynthesis of secondary metabolites. A suspension culture comprises cells and cell aggregates that disperse and develop in a liquid medium that moves. Before the culture hits achieves the maximum cell mass, a part of the culture may be harvested and the fresh medium may be added in order to get a similar growth and performance pattern. However, the cell populations of most long-established cultures display genetic variation, typically resulting in variations of chromosome number or chromosome morphology. It is not possible to eradicate such heterogeneity from such a mixed population of cells (Torres, 1989). Various food additives like anthocyanins, carotenoids, crocin, vanillin, capsaicin, and sweeteners (Curtin et al., 2003; Rhodes et al., 1991; Chen et al., 2003; Dornenburg and Knorr, 1993; Rao and Ravishankar, 1999) and pharmaceuticals like ajmalicine, taxol, morphine, codeine, shikonin, vinblastine, and vincristine (Rao and Ravishankar, 2002) are produced through cell culture. The third aspect of commercial plant tissue culture industry is artificial/synthetic seed production and preservation. The technique is also used for the preservation of endangered plant species. Initially it was recognized as an encapsulated single and somatic embryo as it can generate a whole new plant (Murashige, 1977). Later, other vegetative plant parts like shoot and axillary buds, cell aggregates, and other propagules were also used to produce synthetic seeds if they could germinate like seeds under an ex vitro environment (Ara et al., 2000). Production of artificial seed requires explant (somatic embryo, shoot bud, other propagules), gelling agent (alginate matrix) nutrients and plant growth regulators, and other required chemicals such as bactericides or fungicides (Rihan et al., 2017). Artificial seeds can be produced from nonseed production species, plants with lower germination rates, elite genotypes, hybrid seeds, and genetically engineered plants. Artificial seeds have several advantages, including pathogen-free plants, direct field transfer, genetic uniformity, low cost and faster growth, and germplasm storage (Daud et al., 2008). These seeds can be stored for in the short or long term depending on the method of preparation (Mohamed, 2009). Many protocols have been established from different explants of many plant species, including vegetables, fruits, ornamentals, medicinal plants, field crops, and woody plants. They are thoroughly reviewed by Reddy et al.