The constant agricultural evolution and the high demand from the growing population for food with nutritional value force improvement programs to accelerate the production of improved varieties with desirable agronomic properties. The main purpose is to seek alternatives that lead to the integration of new methodologies and technologies, such as the production of doubled haploid plants (Barro et al., 2001; Achar, 2002).

The doubled haploid plants make up a very useful tool for the breeder in the generation of new and improved varieties (Tuvesson et al., 2000). Currently, one of the main drawbacks of conventional breeding programs for autogamous species is the long period needed to produce an improved line. Due to several generations of self-pollination are required to achieve homozygous genotypes. Once the new genotypes are obtained, the agronomic and industrial traits of interest are consecutively evaluated until they can finally be delivered to farmers (Jacquard et al., 2009).

Biotechnological techniques involving in vitro culture of anthers, isolated microspores, ovaries, etc., can be used with the same efficacy as conventional methods of self-fertilization or backcrossing (Guzy-Wrobelska and Szarejko, 2003). With the in vitro techniques, it is possible to develop resistant cultivars to various plant diseases; saving time, labor, and space in the experimental field (Wędzony et al., 2009). The evaluation of these methodologies will constitute an important tool for the genetic improvement and for obtaining new varieties with excellent agronomic attributes so that they can be distributed to the farmers in less time.

Several reports are concluding that duplicate haploids can accelerate the production of new plant varieties of agronomic interest, being them applied in different species (Thomas et al., 2003). Among the most outstanding crops are barley (Hordeum vulgare L.), pepper (Capsicum annuum L.), rice (Oryza sativa L.), tobacco (Nicotiana tabacum L.), and wheat (Triticum aestivum L) (Ferrie, 2007).

The first report on the use of haploid lines in pepper was published by Christensen and Bamford (1943), finding spontaneous haploid plants among the evaluated material. Similarly, in 1945, Toole and Bamford obtained for the first time doubled haploid plants in cultivars of Capsicum L. using colchicine. Subsequently, Campos and Morgan (1958) succeeded in inducing haploids by intraspecific crossing (Dumas de Vaulx and Pochard, 1974). The yield of these haploid lines was similar to their parents, although with a much lower percentage of fertility; these investigations opened the way to use haploids in plant breeding. The next step in the use of haploids was the development of biotechnological techniques such as the cultivation of anthers, developed in the early seventies. Wang et al. (1973) and George and Narayanaswamy (1973) were the first to report the successful regeneration of haploids in pepper from anther culture (Harn et al., 1975). These researches showed embryonic induction, but a successful regeneration of plants was rarely obtained.

In the works carried out by Sibi et al. (1979) and Dumas de Vaulx et al. (1981) in Capsicum, using the cultivation of anthers, the purpose was to make the technique more efficient in terms of obtaining regenerated plants by making certain modifications. Thus, for instance, the changes made by Sibi et al. (1979) consisted of pre-treatment of anthers in cold (4 °C, 48 h) and determination of the state of optimal development of the microspore (medium or late uninucleate), which allowed them to obtain seedlings for each isolated anther. On the other hand, Dumas de Vaulx et al. (1981) exposed the anthers to thermal shock of 35 °C during eight days of darkness, obtaining between 5-10% of regenerated plants of pepper genotypes.

Dolcet-Sanjuan et al. (1997) describe a new protocol using a biphasic system of semi-solid and liquid medium, replacing sucrose with maltose and enriching the atmosphere of the anther culture medium with CO₂ for androgenesis in pepper hybrids, where they managed to increase the number of embryos (up to 3,561 per 100 fIowers) and plants (up to 23 per 100 fIowers) with 65% of plants duplicated spontaneously. Time later, Yin et al. (2010) confirm that pretreatment at low temperatures, the combinations of growth regulators, the concentrations of activated carbon, and pre-culture temperatures are critical factors that affect the formation of androgenic embryos.

Over the years, research aimed at the use and production of doubled haploids has focused on the refinement of in vitro culture. It has been seen that the optimization of in vitro factors such as quality of the medium, light intensity, temperature, nutrient replacement, among others, improve the androgenic response of the obtained in vitro-plants. However, it has been shown that this response is highly infIuenced by its different genotypes (Morrison et al., 1986).

Despite the advantages offered by the aforementioned processes, there are numerous reports that argue the recalcitrance of the genus Capsicum to in vitro morphogenesis, which manifests itself in various ways such as the low efficiency of the regeneration systems as well as the low reproducibility of the protocols of regeneration -the high index of deformed somatic embryos, the low rate of germination and/or conversion of somatic embryos into plants (Steinitz et al., 2003). Besides the above, a protocol for a specific genotype for the production of doubled haploids has not been fully established (Irikova et al., 2011). The most critical steps are the production of structures similar to the embryos and the subsequent generation of the plant, the collection of fIower buds in the optimal state, stress treatments, in addition to the environment and the culture conditions (Irikova et al., 2011).

There is no specific protocol or culture condition for Capsicum spp. to induce androgenesis in vitro. However, some common guidelines are provided for plant formation from anther culture (Figure 2). In general, most studies report an optimal floral bud size between 3 and 6 cm in diameter. The buds are washed with distilled water, and surface disinfection is performed with calcium hypochlorite, 70% ethanol, Tween 20, among others. After determining the developmental stage of the microspore through staining with acetocarmine, Schiff reagent or DAPI, among others, those in early or late uninucleate phases are cultured in induction media with plant growth regulators (2,4D, dichlorophen- oxyacetic acid; KIN, kinetin, among others) and maintained under a pre-treatment at approximately 28-35 °C with light (12-18 h) and dark (6-12 h) periods. Following embryo formation, these cultures are transferred to regeneration media (free of growth hormones) to stimulate the formation of roots and shoots. The entire process is conducted under aseptic conditions in a laminar flow cabinet (Reinert and Bajaj, 1977). The most used methods for ploidy analysis of in vitro plants are cytological techniques involving chloroplast counts in guard cells, chromosome counts in somatic cells from root apices, as well as flow cytometry. Besides, isoenzyme and DNA marker-based techniques are also used (Germanà, 2011).

Figure 2

Similar to the protocol for anther culture, there is no standard condition to induce plant formation from microspores. The explant can come from microspores or immature pollen to obtain haploid plants, with the advantage that embryos can be obtained from heterozygote somatic tissue (Regner, 1996). This methodology is similar to the one for anther culture, which begins with the mechanic isolation of microspores from a single anther using a glass rod. The microspores are then cultured in an isolation medium containing sucrose, ascorbic acid, L-proline, biotin, and nicotinic acid, according to most studies. The microspore is generally suspended using a nylon net and then transferred to liquid or solid culture media without growth hormones (regeneration or multiplication media). Finally, a haploid plant is obtained and, subsequently, a double haploid plant by treatment with colchicine. This protocol is performed under sterile conditions (Das et al., 2018).

The anther culture technique is often the method of choice by most researchers for the production of doubled haploid. The advantage of this procedure is that the nutritional requirements of the anthers are much simpler than those of the isolated microspores (Bajaj, 1990), and it is not as laborious as microspore culture. Despite these advantages, there are several limitations, such as the regeneration of anther wall somatic tissue, the production of mixoploids (in somatic and gametic tissue), and the formation of asynchronous microspores (Kott et al., 1988). Besides, the use of grains in advanced developmental stages is challenging since these suppress the androgenic capacity of the younger grains by releasing toxic substances (Bhojwani and Razdan, 1996).

Another determining factor for the success of anther culture is the selection of the floral buds since pollen in the early uninucleate phase is more sensitive to external stress, and this could hinder embryogenesis (Irikova et al., 2011). According to Kim et al. (2004), the optimal anther phase for embryo production is the early binucleate phase. Likewise, Supena et al. (2006a) determined that a critical factor for C. annuum L. cultivars is the selection of microspores in the late unicellular phase.

The difficulties discussed can be overcome by culturing isolated microspores. This technique allows not only to discard the use of somatic tissue (i.e., the unknown effect of the anther wall) (Mishra and Goswami, 2014) but also to isolate and culture microspores in an adequate phase; thus, contributing to an efficient regeneration. In particular, there are thousands of microspores per anther, which allows obtaining a greater number of haploid plants, and this can be applied at a large scale on a variety of genotypes (Taşkin et al., 2011). Supena et al. (2006b) found a greater rate of spontaneous re-diploidization from isolated microspores compared to cultured anther. These results prompted studies such as the one by Lantos et al. (2009), in which isolated microspore cultures in pepper genotypes were improved by applying wheat ovary co-culture. Similarly, Heidari-Zefreh et al. (2018) improved the efficacy of microspore embryogenesis and, subsequently, the regeneration frequency of C. annuum L. seedlings by applying different concentrations of ascorbic acid in the medium along with a thermal shock. Despite the success of these studies, there is limited available research regarding isolated microspore culture in C. annuum L., mainly since this technique requires more suitable equipment and technical-scientific skills.

The research focused on the rapid development of improved cultivars using in vitro techniques usually requires first obtaining haploid plants for them to be later diploidized (duplication of chromosomes). Seedlings derived by androgenesis lead to the spontaneous formation of haploids and doubled haploids; however, the percentage of the latter is very low. Due to this fact, it has become necessary to resort to chemical substances, including colchicine (C₂₂H₂₅NO₆), widely used for the induction of polyploidy in plants (Urwin, 2014). This substance is an alkaloid extracted from Colchicum autumnale, which inhibits the self-assembly of tubulin, preventing the formation of the microtubules of the spindle, affecting only cells that are in the division, therefore acting as a “mitotic poison” (Badu et al., 2017). The most important advantage of polyploidy is that plants tend to have better performance and morphological characteristics, such as the height and size of plant organs (Hannweg et al., 2016), and the increase in biomass in general (Urwin, 2014).

This mutagenic can be applied to plants in different ways: by aqueous solution submerging the roots (systemic absorption) or by microinjection, in which the solution is incorporated into the in vitro plant by mechanical inoculation. On the other hand, calli can also be imbibed in colchicine before they are planted in the regeneration medium (Dhooghe et al., 2011).

A large number of polyploid induction processes have been reported with colchicine, being a very effective mechanism, but it has also been seen that its use can generate chimerization and death of the tissue with which it has been in contact, so the production of viable polyploid plants decreases in a large proportion (do Rêgo et al., 2009). Because colchicine has negative effects, the use of Oryzalin and other methods that affect mitotic processes, have begun to be used, generating higher rates of viable polyploid seedlings.

Techniques for the chromosomal duplication of haploids in pepper cultivars are generally based on treatment with colchicine. Gémesné Juhász et al. (2001a, 2001b) showed that they could increase the efficiency in 95% of doubled haploid production in pepper cultivars (Capsicum annum L.) by applying colchicine (0.04%) to the regeneration medium. In subsequent investigations, Gémesné Juhász et al. (2001a) indicated that the medium supplemented with colchicine resulted in an effective diploidization of 50-95% explants.