**1. Introduction**

Due to the growing global crisis caused by climate change and greenhouse gases, which are significantly affecting food availability and price volatility in the agricultural sector, agricultural systems have been forced to implement new adaptive measures with the aim of developing alternatives that improve and accelerate traditional methods of food production through biotechnology [1, 2].

Plant biotechnology had an important development in the last decades, moving from soil cultivation to practices where ecological factors are involved with sustainable objectives such as water conservation, decrease of soil erosion, and higher yields and better-quality crops. As a result of this movement, the technique of in vitro

culture arose, in which plants are propagated inside containers under laboratory conditions [3] and has open the possibility of enhancing two conditions in agricultural crops, firstly, the asepsis, which is the absence of germs, and secondly, the control of factors that affect plants growth.

However, although it is a technique that has been widely used in agricultural development and plant research, the in vitro culture technique is more expensive than traditional methods of plant production, due to the components used for the preparation of the culture medium, commonly the gelling agent called agar [4–6].

The culture media must meet the nutritional requirements necessary for the development of the plant and these depend on the culture used, which is in some cases solidified to provide support to the explant through the gelling agent agar. Agar is a mixture of polysaccharides extracted from the walls of red algae (Gelidium, Gracilaria, Pterocladia, Gelidiella) and is the most widely used gelling agent in culture media due to its physicochemical properties such as porosity, thermo-reversibility, stability, gel strength, texture, elasticity, and transparency. Its use increases the total cost of the medium by 70%, due to agar overexploitation and high demand [7].

As a result of this, recent studies have made it possible to know with greater precision the availability of alternative substances to agar in naturally occurring raw materials that are easier to extract and have the same characteristics as agar. The study of natural polymers of vegetable origin has emerged as a sustainable alternative due to their properties. Starches and gums have been investigated due to their high availability in local markets and low cost [8–10], concluding that they can potentially replace agar partially and/or totally as gelling agents due to their physicochemical composition, and can reduce costs of this technology due to their greater efficiency, ease of extraction and acquisition [11].

The nopal cactus (Opuntia ficus-indica), a native plant distributed throughout Latin America, is a natural polymers supplier that has been studied in numerous investigations for its properties, since phenolic compounds had been found in the composition, which can be used in various industries, including pharmaceutical, construction, agrotechnology, bioenergy, and biotechnology industries [12]. One of the most important components of nopal cactus is dietary fiber, which is a rich source of hydrocolloids (pectin and mucilage), named as such because of their great capacity to capture and retain water.

Pectin is mainly composed of galacturonic acid, and mucilage is composed of arabinose, galactose, xylose, rhamnose, and galacturonic acid (classified as an acid mucilage) [13]. These are hydrocolloids that have the ability to gel and form gels in combination with the indicated solution. In addition, it is well known that one of the main functions of mucilages is to promote seed germination since when they come into contact with water, they increase their volume, forming a moist layer around the seed, which facilitates germination and protects the plant from external damage [14, 15].

Recently, nopal cactus hydrocolloids have been used as a thickening, stabilizing, encapsulating, and moisturizing agents in different research projects; however, in this chapter we will focus on the study of the nopal cactus hydrocolloids for their potential to substitute (partially or fully) the gelling agent agar for in vitro culture media applications, benefiting from the molecular structure and the mechanical properties of those compounds.

First, we will describe in vitro culture and the important features for the development of this technique, followed by the gelling agents and hydrocolloids mostly used to improve the technique and the recently studied substitutes, including their characteristics. Finally, we will discuss the potential application of nopal cactus

hydrocolloids as gelling agents in in vitro culture and describe their molecular structure and mechanical properties.

### **2. The history of in vitro culture**

The term "in vitro tissue culture" means growing explants in a glass bottle in an artificial environment in which asepsis, growth, and development must be controlled. Plant culture has undergone a significant evolution in the last 100 years. The knowledge and research related to cell theory has reached a significant development, which has generated a disruptive innovation in traditional culture.

The history of in vitro culture began in 1887 with Schleiden and Schwan, who explained that a cell could subsist by itself if the external conditions were favorable for its growth (cell theory). A decade later, in 1902 Herbertland, pioneer of in vitro culture and called the father of the technique, said that plants were capable of reproducing their growth from isolated cells. Herbertland proposed that it was possible to grow free vegetative cells and pollen tubules together by adding nutrient solutions supplemented with extracts of vegetative apices or with fluids from embryo sacs, and although he could not prove it due to the simplicity of his culture media, it was a breakthrough that gave the guideline to start investigating the technique [16].

In addition, Herberlat was the first to mention cell totipotency, which is defined as the ability of a cell to generate an individual completely identical to the mother cell from a single cell, which contains the same genetic material of the plant to which it belongs, therefore, it has the potential to generate a completely new plant [17]. By 1924, Blumenthal and Meyer demonstrated callus formation using carrot slices and lactic acid.

In the 1930's White, Gautheret and Nobécourt decisively demonstrated the possibility of cultivating plant cells in vitro, discovering two important characteristics, the identification of auxins as regulators of plant growth and the importance of B complex vitamins in plant growth [18]. By 1940 Blakeslee, Conklin, and Van Overbeek studied a semisynthetic liquid medium with coconut milk, which had a good proliferation response, which promoted biochemical research.

In 1962 Murashige and Skoog made a medium for tobacco tissues that contained all the nutrients necessary for the growth and proliferation of cays, and a high concentration of salts that benefited the growth of somatic embryos [19]. Subsequently, research was carried on somatic embryogenesis, explant types, the culture of microspores and meristems, and obtaining hybrids.

Culture media had been an important object of study, in 1984, Wetherell proposed that high levels of sucrose up to 12%, nitrate, and iron are essential for somatic embryos to develop to maturity [20]. However, nowadays most culture media consist of five groups of ingredients: organic nutrients, carbon source, vitamins, and growth regulators such as phytohormones and their inhibitors (auxins, gibberellins, cytokinins, abscisic acid, ethylene, brassinosteroids, polyamines, jasmonic acid, and salicylic acid).

Auxins promote cell elongation and cytokinins stimulate cell division [20]. These media are mostly used to promote organogenesis and their composition is determinant for growth. The Murashige & Skoog (MS) medium designed for tobacoo cells is the most used due to its success for agar culture [21] although there are other media such as Schen and Hildebrandt, Heller, MS, and Eriksson, among others, which contain certain macro and micronutrient content that are also used for plant cell culture.

Another factor that influences the growth of tissues in solid culture media is their consistency, which is the result of the selection of the gelling agent [22]. There is a great variety of gelling agents, the most used are agar, agarose, gellan gum, or calcium alginate [23]. Agar is commonly used in proportions of 0.6% to 1.0%, due to its composition, purity, and properties, which do not interfere with the growth of the culture [24].
