Polyploidization is a slow and gradual process that has driven plant evolution and speciation over time. However, artificial polyploidization can induce polyploidy in a shorter period (Eng & Ho, 2019). There are several methods of inducing polyploidy in plants, such as inducing polyploidy in somatic cells by the disrupting chromosome disjunction at anaphase of mitosis (Trojak-Goluch et al., 2021).
Colchicine, an alkaloid extracted from the seeds and roots of autumn crocus (Colchicum autumnale L.), is the most effective and widely used antimitotic agent to induce polyploidy in medicinal plants (Adaniya and Shirai 2001; Rubuluza et al. 2007; Sadat et al. 2011; Widoretno 2016; Noori et al., 2017; Ahmadi et al., 2021; Purbiya et al., 2021; Tsai et al., 2021). Colchicine dissolves well in alcohol and chloroform, less well in water and glycerol, and has multiple properties and a complex mechanism of action (Karamanou et al., 2018). Its action disrupts the function of microtubules responsible for the propagation of chromosomes to daughter cells, leading to the formation of polyploid nuclei (Manzoor et al., 2019).
Inducing chromosome doubling involves a series of steps, including an induction phase, growth phase, and a confirmation technique to evaluate the rate of achievement. The induction phase depends on different factors, such as method type, type of plant tissue, antimitotic agents, their different concentrations, and exposure durations. In cases of in vivo treatment, the antimitotic agent is generally applied through syringe injection, foliar spray, or cotton plug method. Another method for inducing polyploidy is soaking seeds and plunging the roots in aqueous solutions of mitotic inhibitors (Salma et al., 2017). The colchicine concentration and treatment duration are interdependant and crucial in polyploidization induction. The colchicine concentration applied ranges from 0.05–1.0% and duration of treatment ranges from hours to weeks, depending on the method of application. Higher concentrations and longer colchicine treatment often result in higher lethality of explants (Tavan et al., 2015; Zhou et al., 2017).
High concentrations are toxic to plant cells and usually cause abnormal plant growth or reduced viability, while low concentrations may be ineffective or induce many mixoploids or aneuploids (Manzoor et al., 2019). There are two ways to identify polyploids, namely indirect identification and direct identification. Most polyploidization studies conduct indirect identification first, followed by direct identification to accurately identify polyploids (Moghbel et al., 2015). Indirect methods are easy, less time-consuming, and use simple instruments for screening (Eng & Ho, 2019). Different morphological and physiological traits, particularly pollen diameter, number of chloroplasts, stomatal size, and stomatal density, can be studied through these indirect methods (Moghbel et al., 2015). The measurement of stomatal dimensions is effortless and cost-effective (Salma et al., 2017). In direct methods, techniques like chromosome counting have been examined as an effective and reliable method, but it is a laborious process, particularly for plant species with highly dense cytoplasm consisting of a large number of chromosomes (Sakhanokho et al., 2015). Moreover, a specific protocol is required for each plant species. Thus, flow cytometry (as an indirect method) is considered to be a more reliable, rapid and simple method to analyze a large number of samples in a very short time period (Sattler et al., 2016). Combination of chromosome counting and flow cytometry analysis provides an overall picture of chromosome composition of the cells (Ochatt et al., 2011, Sign., 2017). Based on Eng & Ho (2019) findings, the most commonly used method to determine chromosome number is flow cytometry analysis (70.0%), followed by chromosome counting procedure (56.7%). 46.7% of studies used combination of both methods. Previous studies have established polyploidization protocols in several medicinal and aromatic herbs, although the protocols are rather species-specific and may not be effective on other species. Thus, proper species-specific method development for polyploidization is a prerequisite to achieving polyploids effectively (Julião et al., 2020). Successful application of the in vivo system for induced polyploidy has been reported in several studies, including Gossypium arboreum (Yang et al., 2015), Jatropha curcas (Chiangmai et al., 2014), Eribotrya japonica (Blasco et al., 2015), Stevia rebaudiana (Zhang et al., 2018), Carum copticum (Akbari et al., 2021), Trachyspermum ammi (Dwivedi and Kumar, 2021) and etc.
Some researchers have reported very low numbers of polyploids in certain plant species, such as 3.7% in Centella asiatica (Kaensaksiri et al. 2011), 4% in Salvia hains (Sadat et al. 2011), and 5% in Echinacea purpurea (Abdoli et al. 2013). However, other studies have reported much higher percentage of tetraploids, such as 40% in Scutellaria baicalensis (Gao et al. 2002), 66.2% in Pfaffia glomerata (Gomes et al. 2014), and 100% in Pogostemon cablin (Widoretno 2016).
Cumin (Cuminum cyminum Linn.), the king of seed spices (Lal et al.,2014), which belongs to the Apiaceae family, is highly valued for its aroma, medicinal properties, and therapeutic benefits, even in long-time stored seeds (Sowbhagya,2013). It is the second most popular spice in the world, after black pepper (Lodha and Mawar,2014), and is widely used in the food, cosmetics, and perfume industries (Dubey et al., 2017). Despite its economic and medicinal importance, no established protocol for polyploidization in C. cyminum has been reported to date.
Here, we describe a successful method for producing synthetic polyploid plants of C.cyminum using colchicine. The results of our study provide a foundation for future breeding efforts aimed at enhancing essential oil yields in medicinal and aromatic herbs through polyploidization. Additionally, our protocol may serve as an optimized approach for artificial polyploidization in C.cyminum and related species, leading to the development of improved genotypes.