Garlic (Allium sativum L.; family Alliaceae; 2n = 2x = 16, is a native of Central Asia and is one of the oldest crops grown throughout the world for food. Garlic contains water (65%), carbohydrates (26-30%), and other components such as proteins, lipids, fiber, minerals, and saponins. Garlic also contains phenols, vitamins A, C, E, and B-complex vitamins (thiamin, riboflavin, niacin) and other elements such as selenium, sulfur, zinc, magnesium, iron, sodium, calcium. Garlic has a high nutritive compound and several medicinal values such as antibacterial, antifungal, antiviral, antioxidant, and anticancer properties (Arisuryanti et al. 2018).

The production of garlic has not been able to meet the demand for food consumption in many countries of the world, causing a considerable gap between consumption and domestic production. The production of garlic is limited by viral diseases, high production costs, high garlic consumption demand, less competency in producing garlic with large bulb size (Nur 2018). Commercial types of garlic are divided into four categories such as violet or Asian, which is cultivated in subtropical regions, pink, which needs long photoperiods and has low requirements for cold, white, which needs long photoperiods, has medium to high requirements for cold, and purple, which needs long photoperiods and periods of cold (Sinha et al. 2016). Garlic is also classified into hard-neck and soft-neck structures based on the genotype of garlic varieties. Garlic varieties with hard-neck form a floral scape where their flowers abort easily, while the garlic varieties with soft-necked do not form a scape. However, most of the commercially cultivated garlic species possess soft-necks because they are easier to cultivate and have longer shelf lives.

Vegetative propagation of garlic gives low genetic variation and multiplication rate in the field. Due to the floral organ abortion and chromosomal abnormalities in garlic, pollination is difficult to happen naturally. Garlic breeding mainly depends on clonal selection and mutation breeding. Colchicine is the most effective chemical mutagen senso latu in inducing ploidy levels in several garlic species (Spencer-Lopes et al. 2018). Colchicine affects chromosome doubling on mitotic cells by binding to the positive end of microtublin leading to the disruption of microtubulin polymerization (Roughani et al. 2017). The spindle fibers are used to pull the sister chromatids to opposite poles of the cell. However, without the action of spindle fibers, the mitotic process could be disrupted. This disruption results in DNA replication without cell division (Roughani et al. 2017). Polyploid plants possess larger seeds and morphology than diploid plants because of their larger cell size (Münzbergová 2017). Polyploid plants induced by colchicine in vitro results in an increase in DNA content and affect the epidermal surface cells. Polyploids have a thicker cell size but have a low cell number per leaf blade as compared to the diploids (Corneillie et al. 2019).

The significance of this review aims to describe the effect of plant growth regulators in callus production, cell proliferation, regeneration, and colchicine treatment for polyploidy induction in vitro. Improving the genetic potential of garlic by colchicine helps to increase the qualitative and quantitative traits in vitro. In vitro micro-propagation is the best alternative to meet the needs of garlic genetic variability, and has a potential for rapid multiplication and production of high quality, uniform, and disease-free planting material of garlic. Bulb treatment by colchicine can produce a high level of chromosomal mutation leading to the creation of new variants compared to the diploid control. New genetic variations generated by colchicine treatment is particularly advantageous in improving an outstanding genotype. Genetic changes are possible in garlic via colchicine. Colchicine-induction on ploidy increment and its effects on chromosomal changes in garlic is very important to get useful variants for agronomic traits.

The in vitro plant culture technique is an efficient method for clonal propagation of garlic plants from a small tissue fragment in vitro. It is widely used in commercial production and is a rapid method of multiplication that only requires little space in laboratory conditions (Kirilova et al. 2019). Plant tissue culture is a technique in which small tissue or organ pieces (explants) are isolated from intact plants and cultured aseptically on a suitable nutrient medium. The growth and development of the isolated plant part is controlled by manipulating the composition of the nutrient media and environmental conditions during in vitro culture (Khan et al. 2017; Li et al. 2019). The in vitro culture method is used to produce identical progeny or to induce genetic variability. Genetic changes can be performed in vitro culture either by somaclonal variation or the induction of mutagens. In the Allium improvement program, in vitro techniques combined with conventional methods are used for mass clonal propagation of selected genotypes, production of disease-free clones, germplasm conservation, and development of new varieties via cellular of molecular genetics (Khan et al. 2017).

Traditional vegetative propagation has a low coefficient of multiplication and transmission of diseases such as viruses (Khan et al. 2017). Tissue culture techniques have several advantages over traditional propagation methods. Cultures start with small parts of the plant; therefore, only a small amount of space is required to propagate large numbers of plants. In vitro techniques are also free from plants’ viral diseases. However, in vitro culture techniques require specialized facilities and advanced skills for their operation, and the cost of propagation is still relatively high and is frequently associated with hyperhydricity or vitrification. Vitrification is a physiological disorder caused by the condition of in vitro culture and promotes the physiological, anatomical, and morphological abnormality of plants such as slow growth rate, thick and deformed stems with translucent, thick, and wet leaves and shoots.

Sterilization

The duration of explant sterilization varies widely from 5 minutes to several hours in Allium species due to the differences in seed surface structure and the extent of surface contamination (Fenwick et al. 1985). In sink organs (bulb or cloves), it is better to sterilize until they have good contact with all surfaces of the plant tissues. In heavily contaminated plant parts such as bulbs dug from the soil, repeated sterilization is recommended to protect from microorganisms. Garlic plants can have fewer superficial contaminants if cloves are treated before planting with the systemic fungicide. Growing plants should be kept in relatively low humidity for 4 to 6 weeks before explant excision (Haim et al. 1990). Allium tissues have also internal infections of non-pathogenic microorganisms. The eradication of such microorganisms is much more difficult than those on the surface. Some contamination problems may be overcome by using antibiotics. However, Allium organs, especially leaf tissues, are sensitive to penicillin, streptomycin, and gentamycin and show variable responses according to the genotype and physiological status of the plant. Dithane and Agrimycin are common chemicals used in garlic sterilization in vitro at the rate of 2.0 mg/L each for 16-20 hours in vitro (Hailu et al. 2020). Dithane (Mancozeb) is a systemic chemical used to control fungi while Agrimycin (Oxytetracycline) is used to control bacteria in vitro tissue culture.

Garlic micro-propagation in vitro

Most cultivated Allium species are propagated by seeds. Seeds have several natural advantages as a means of propagation. Plants propagated by seeds may be stored for long periods without the loss of viability and is possible to produce in large numbers so that the plants grown from them are individually inexpensive (Haim et al. 1990). However, in plants that do not produce viable seeds, vegetative propagation may be necessary for breeding programs in seed propagated Allium species. Garlic is propagated asexually from cloves because of the sterile flowers, which are totally or partly replaced by bulbils (Cheng et al. 2012). Virus-free clones produced via meristem culture could have higher yields with better quality (Luciani et al. 2006; Haider et al. 2015). Stem disc or clove base is an effective organ in callus or adventitious bud induction (Zhang et al. 2016). Improvement of garlic production can be achieved through chromosomal mani-pulation and selecting desirable variants such as large bulb size, number of cloves, storability, etc. Sexual propagation of garlic is highly impossible due to the failure of fertile seed production, hence garlic in cultivation is propagated asexually by planting individual cloves (Sinha et al. 2016). Vegetative propagation of garlic offers limited chances for creating genetic variation and gives a low multiplication rate in the field (Sinha et al. 2016). Garlic is inefficient in multiplication because of its sexual sterility, likely for viral disease transmission. Garlic is propagated through vegetative means and the improvement of garlic through breeding programs is limited due to difficulties of inducing flowering (Mubarrat et al. 2018).

Garlic is one of the several important vegetable crops within the Allium family and is widely grown for its culinary and medicinal properties. It is propagated through vegetative means due to non-fertile flowers (Khan et al. 2017). In vitro regeneration of plants depends on several factors such as the explant type and its physiological condition, genotype, and growth regulator combination used in the culture medium (Luciani et al. 2006; Hassan et al. 2014; Mubarrat et al. 2018). It is very susceptible to viral, nematodes, and fungal diseases with low propagation efficiency in the field. Plant tissue culture is used for the production of pathogen-free plants and the multiplication and conservation of vegetatively propagated or plants having unviable seeds (Khan et al. 2017). Plants regenerated from meristematic tissues, such as shoot-tips or axillary buds are commonly true to type and genetically uniform. If maintenance of the clones of garlic plants is needed, meristem tip, flower head, basal plate culture systems should be used. In vitro induction method is useful because of its low cost to apply, easy to operate, useful to replicate the experiment and it has a controlled environment (Li et al. 2019).

Explant selection for mutagenic treatment

Before inducing a mutagenesis program, different considerations should be taken such as the efficiency of mutagenesis, the explant, and mutant screening (Hase et al. 2012). Mutations are performed by exposing the plant propagules to physical and chemical mutagenic agents. In vegetatively propagated plants, plant parts such as stem cuttings, twigs, buds, and tubers can be exposed to mutagens, while seeds are used in the case of seed-propagated crops (Bahadur et al. 2015). The parental material used for mutagenesis should be free of con-tamination. The type and concentration of mutagen to be used for mutagenesis may vary depending upon the type of material chosen. The plants’ tissues that are metabolically active or those that have higher water content are more sensitive to mutagens. In vitro cultures are used for mutation induction for vegetatively propagated crops (VPCs) (Suprasanna et al. 2012).

The concentration of mutagen

The concentration of the mutagens should be sufficiently high to increase the probability of inducing mutation. However, it should not be so high as it may cause lethality to the cells/tissues. LC50 (lethal concentration 50%) is used to determine the optimum concentration to be used in the mutagenesis experiment. It causes 50% lethality in the plant organism used for irradiation at a defined time. LC50 varies with the plant species, the type and status of the material, and the stage where the lethality is measured (Bahadur et al. 2015). The concentration of chemical mutagen is determined based on the properties of the mutagen (half-life, penetrability, solubility, toxicity, or reactivity); type and condition of the treated material before, during, and after treatment; interaction with target tissue and culture medium; pH of the growth medium; and post-treatment handling of the material.

Meristem-tip culture

Meristem-tip culture is commonly used in vitro methods for clonal propagation and virus eradication in Allium species (Luciani et al. 2006; Robledo-Paz and Manuel 2012; Haider et al. 2015). Meristem culture maintains high genetic stability and is used for in vitro germplasm conservation (Haider et al. 2015). Meristem-tip culture of garlic is commonly applied for virus eradication and/or micro-propagation (Luciani et al. 2006; Haider et al. 2015). For virus elimination, it is recommended to isolate a meristematic dome with one leaf primordium which is shorter than 5 mm (Robledo-Paz and Manuel 2012). Meristem-tip culture has been used for experimental mutagenesis and polyploidy induction in garlic (Gantait et al. 2011). Isolated meristem-tip culture seems are more suitable than the intact plant for mutation and polyploid breeding of vegetatively propagated Allium species. Meristem-tip culture is used for the production of identical plants (micro-propagation) or to induce genetic variability (Sofia 2007; Gantait et al. 2011; Kong et al. 2014).

Growing medium selection

Growth media should be optimized for the period of induction. The period starts with a high concentration of plant growth regulators (PGRs), but gradually declines with time due to degradation (caused by lability at growth temperatures, light) and uptake (Trigiano and Gray 2004). The addition of activated charcoal to culture media alters the effective concentration of PGRs due to adsorption. In micropropagation of Allium spp., regeneration and multiplication of plantlets are affected by the composition of the culture medium, combination of growth regulators used, and the kind of explants. Basal Dunstan and Short (BDS) medium improves the growth rate of garlic callus by adjusting nutritional and hormonal ratios (Table 1) (Luciani et al. 2001; Luciani et al. 2006). Basal Dunstan Short (BDS) is a modification of B5 medium which contains NH₄H₂PO₄ and NH₄NO₃ added to increase PO₄H₂⁻, NO₃⁻ and NH₄^＋ levels, thus changing NO₃⁻/NH₄^＋ ratios (Luciani et al. 2001; Gantait et al. 2011). All types of Allium tissue cultures can be possible to culture on Murashige and Skoog (MS) medium (Table 2), sometimes half-strength (Murashige and Skoog 1962). However, some studies also used Gamborg’s B5 medium.

Table 1 . Composition of inorganic salt stalks in Basal Dunstan Short (BDS, 1977).

Chemical compound	Conc. (g/L)	Vol. (ml/L)	Molarity
Calcium chloride (CaCl2·2H2O)	30	20	1.02 mM
Ammonium nitrate (NH4NO3)	126.5	20	25.02 mM
Potassium nitrate (KNO3)	16.008	20	4.0 mM
NH4H2PO4	0.230	20	2.0 mM
(NH4)2 SO4	0.134		1.01 mM
Magnesium sulfate (MgSO4·7H2O)	49.4	20	1.00 mM
Manganous sulfate (MnSO4·4H2O)	2.64		0.045 mM
Zinc sulfate (ZnSO4·7H2O)	0.4		6.95 mM
Cupric sulfate (CuSO4·5H2O)	0.0078		0.1 mM
KI (Potassium iodide)	0.15	20	4.52 mM
Cobalt chloride (CoCl2·6H2O)	0.005		0.105 mM
Boric acid (H3BO3)	0.6		0.049 mM
Sodium molybdate (Na2MoO4·2H2O)	0.05		1.03 mM
NaH2PO4.2H20	0.172		1.04 mM
Ferrous sulfate (FeSO2·7H2O)	0.028	10	0.10 mM
Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA.2H2O)	37.25		0.10 mM
Nicotinic acid	0.1	20	8.1 mM
Thiamine HCl	0.001		0.03 mM
Pyridoxine HCl	0.1		4.9 mM
Meso-Inositol	10	10	1.8 mM
Sucrose	30		0.088 M
2,4-D (2,4-dichlorophenoxyacetic acid)	0.4 mg/L		1.25 mM

Table 2 . Composition of inorganic salt stalks of the Murashige and Skoog (1962).

Chemical compound	Conc. (g/L)	Vol. (ml/L)	ppm (mg/L)
Ammonium nitrate (NH4NO3)	82.5	20	1,650
Potassium nitrate (KNO3)	95	20	1,900
Magnesium sulfate (MgSO4·7H2O)	74	5	370
Manganous sulfate (MnSO4·H2O)	4.46		22.3
Zinc sulfate (ZnSO4·4H2O)	1.72		8.6
Cupric sulfate (CuSO4·7H2O)	0.005		0.025
Calcium chloride (CaCl2·6H2O)	88	5	440
Potassium iodide (Kl)	0.166		0.83
Cobalt chloride (CoCl2·2H2O)	0.005		0.025
Potassium phosphate (KH2PO4)	34	5	170
Boric acid (H3BO3)	1.24		6.2
Sodium molybdate (Na2MoO4·2H2O)	0.05		0.25
Ferrous sulfate (FeSO2·7H2O)	2.78		13.9
Ethylenediaminetetraacetic acid, disodium salt (Na2EDTA.2H2O)	3.73		18.65
Myo-Inositol	10	10	100
Thiamine	0.01	10	0.1
Nicotine	0.05		0.5
Pyridoxine	0.05		0.5
Glycine	0.2		2.0
Sugar	30

According to Sinha et al. (2016), explants cultured on Murashige and Skoog (MS) medium supplemented with BAP (Benzylaminopurine) and Kinetin at various con-centrations ranging from 1.0-5.0 mg/L either alone or in combination are suitable for garlic proliferation. An optimal concentration of Kinetin and BAP (Benzylaminopurine) are used for inducing polyploidization. The use of meristems as initial explants and 2,4-D (2,4-dichlorophenoxyacetic acid) as the appropriate growth regulator can enhance callus induction (Luciani et al. 2006). Khan et al. (2004) and Haque et al. (2003) reported Murashige and Skoog (MS) medium supplemented with zero concentration of 2,4-D (2,4-dichlorophenoxyacetic acid) has no response for callus induction while Murashige and Skoog (MS) medium without any growth regulator is best for root induction in garlic. Murashige and Skoog (MS) medium supplemented with different concentrations of BAP (Benzylaminopurine) show a wide variation in shoot proliferation of garlic variety (Khan et al. 2004). Kinetin is best for shoot-root initiation and proliferation (Haque et al. 2003). Luciani et al. (2006) reported auxins to induce callus formation, proliferation, and somatic embryogenesis while cytokinins induce shoot and root differentiation and elongation. The use of meristematic explants and 2,4-D (2,4-dichlorophenoxyacetic acid) as a growth regulator enhances callus induction and growth-producing friable callus to regenerate plants either by embryo or shoot formation. Gantait et al. (2011) reported the addition of cytokinin at a lower level along with higher concentration of 2,4-D (2,4-dichlorophenoxyacetic acid) enhances callus induction than 2,4-D (2,4-dichlorophenoxyacetic acid) alone. Callus differentiation and plant development are determined by growth regulators (Luciani et al. 2006; Haider et al. 2015). An efficient system for fast production of callus and subsequent regeneration of garlic is a problem in in-vitro culture (Kapoor et al. 2011). Haque et al. (2003), Mubarrat et al. (2018) reported that growth regulators like 2,4-D (2,4-dichlorophenoxyacetic acid) and Kinetin are effective for callus induction. Those hormones play a vital role in vitro regeneration. 2,4-D (2,4-dichlorophenoxyacetic acid), and Kinetin are key hormones that lead to better performance in garlic regeneration. Haque et al. (2003) also reported Murashige and Skoog (MS) supplemented with Kinetin gives high regeneration of somatic embryo and yielded a high number and length of shoots from each callus. Haider et al. (2015) reported a higher concentration of auxin (2.0 mg/L 2,4-D) with (0.5 mg/L) of cytokinin are suitable for the proliferation of callus than that of a lower concentration of auxin while Yanmaz et al. (2010) reported lower auxin and cytokinin concentrations are effective hormones for shoot induction. The essential function of auxins and cytokinins is to reprogram somatic cells that had previously been in a determined state of differentiation. Reprogramming causes dedifferentiation and redifferenti-ation into a new developmental pathway (Trigiano and Gray 2004). High concentrations of exogenous auxin is toxic as they stimulate the production of ethylene, which can cause growth inhibition (Trigiano and Gray 2004; Ayu et al. 2019).

Chemical structure of 2,4-D (2,4-dichlorophenoxyacetic acid) and Kinetin.

Somatic embryogenesis

Somatic or non-zygotic embryogenesis is a process of inducing plant somatic cells to form somatic embryos (Trigiano and Gray 2004). Callus plays a vital role in plant growth and development in vitro (Piršelová and Matušíková 2013). Callus develops embryo (by embryogenesis process), shoot, and root (by organogenesis process) under in-vitro conditions (Kirilova et al. 2019). During the organogenesis, the shoot and root development lead to the generation of the unipolar structure where the vascular system is connected to the maternal tissue (Sang et al. 2016). The formation of structures called embryoids in garlic was reported for the first time in 1977 from calli obtained from stem tips, bulb leaf discs cultured in kinetin, and IAA (Robledo-Paz and Manuel 2012). Embryo development is essential for the production of genetic variability and to improve different plant properties such as stress conditions (Lämke and Bäurle 2017). However, it is difficult to obtain plant embryos in vitro conditions due to the need for special medium composition for callus culture and embryo formation (Guruprasad et al. 2016). In each plant species, there may be different genotypes that can have different embryogenesis capacity due to the difference in response to hormones (Kirilova et al. 2019).

Garlic regeneration in vitro

In Allium micro-propagation techniques, the differentiated shoots require changes in media composition and also in the culture environment to induce roots. Garlic plantlets can induce roots at 25℃ and under 16-hour photoperiods onto the half-strength Murashige and Skoog (MS) medium with decreased sucrose content (10 g/L) and with the addition of 5 P-indolebutyric acid (IBA) (Haim et al. 1990). Garlic explants from meristem basal disc can be cultured aseptically on nutrient media supplemented with growth regulators to produce an unorganized callus tissue. The callus initiation, multiplication, and morphogenesis to regenerate into a whole plant depends on the plant growth conditions, genotype, type, and ontogenetical status of explant taken, combination and levels of growth regulators in the growth medium, and the environmental conditions in which the cultures are kept (Bhojwani and Razdan 1986; Luciani et al. 2006; Hassan et al. 2014; Mubarrat et al. 2018). The physiological state of plant tissue determines the biological response in tissue culture. Certain plant parts (explants) are more responsive than others at a given time. The cotyledons (seed leaves) of some species regenerate shoots well, whereas the true leaves or stems do not. But, in some cases, the physiological state of explants can be altered by pretreatments with another PGR (Trigiano and Gray 2004).

Basal Dunstan Short (BDS) with increased levels of ammonium phosphate and nitrate salts in B5 basal medium, results in more rapid and friable callus growth. Basal Dunstan Short (BDS) is a suitable medium for callus cultures derived from different parts of Allium species (Dunstan and Short 1977). Auxins have multiple roles based on their chemical structure, concentration, and the plant tissue being affected. Auxins stimulate the production of callus, roots, an extension of stem growth, cell elongation, cell division in cambium tissue, differentiation of phloem, and xylem (Trigiano and Gray 2004). Callus induction and growth in subcultures is dependent on the presence of exogenous auxins. 2,4-dichlorophenoxyacetic acid (2,4-D) is a very effective growth hormone for callus induction and growth in garlic. The presence of cytokinins in nutrient media is not usually necessary, but in combination with auxins can result in callus formation (Havranek and Novák 1973). Media and growth regulators that are suitable for rapid cell proliferation and callus formation are not favorable to the initiation of morphogenetic meristems which give rise to shoots and roots. To induce organogenesis, an unorganized callus initiated on a primary medium should be transferred to another medium that contains a different hormonal composition. Garlic organogenesis from callus tissue is usually inhibited by high concentrations of 2,4-D (2,4-dichlorophenoxyacetic acid). To have shoot regene-ration, transfer of callus tissue to 2,4-D free media, or media with a low concentration (up to 0.2 mg/L) of 2,4-D supplemented with cytokinins (kinetin, 2ip, or BAP) is essential (Novak 1986). A hormone-free medium is suitable for inducing shoot regeneration from the callus culture of garlic. Repeated subculture on organogenesis-induction-medium promotes shoot regeneration on garlic. However, calli more than a year old usually fail to regenerate shoots. Root growth takes place on media supplemented with auxin (e.g., IBA). In substitution of IBA, GA₃ also results in extensive root formation (Novak 1986).

Colchicine treatment

In vivo and in vitro colchicine treatments are used to artificially produce polyploids. Mutation breeding by colchicine has been pivotal in increasing the genetic diversity of crops, thereby enriching the genetic pool (Shimelis and Laing 2012). Mutation is a source of variability in garlic; however, breeding using mutation has not resulted in significant progress due to the limitation in variability (Rabinowitch and Currah 2002). Chemical mutagens result in high mutation rates and mostly result in point mutations (Bahadur et al. 2015). Chemical mutagenesis plays an important role in germplasm collection (Kong et al. 2014). Chemical mutagens cause higher genetic changes and smaller chromosomal aberrations compared to gamma and X-rays (Cvejić et al. 2014). The production of a great number of recessive genes and cytoplasmic mutations are the most important effects of both physical and chemical mutagens (Cvejić et al. 2014). Colchicine has been used for a long time as a polyploidizing agent. Colchicine is an effective method for producing polyploid plants, but the response to colchicine differ depending on the plant species (Heo et al. 2016; Bahadur et al. 2015). It has been used successfully to produce polyploids for cytogenetic research and breeding program.

Colchicine is an alkaloid and secondary metabolite extracted from the plant Colchicum autumnale which is the most widely used doubling agent either in garlic or other species (Alan et al. 2004; Alan et al. 2007) with an average doubling efficiency of 70% (Nowak 2000; Jakše and Bohanec 2003). Colchicine is a chemical mutagen senso latu, as its main effect is on ploidy and not on genes (Spencer-Lopes et al. 2018). Appropriate colchicine concentration and immersion duration can stimulate polyploidy in plants. Kong et al. (2014), reported that the rate of plantlet regeneration decreases at higher colchicine concentration and longer time duration. Higher colchicine concentration and longer immersion time decrease the survival rate and root formation in Allium species (Heo et al. 2016). The concentration of colchicine has a stronger effect on plantlet regeneration than time duration (Kong et al. 2014). Too low or high concentration of colchicine with inappropriate immersion cannot stimulate polyploidy or can damage the chromosomes (Khikmah and Suratsih 2019). Colchicine causes ploidation in meristematic cells (Istiqomah and Shofi 2018). Colchicine concentration and duration of treatment are key factors for each type of explants (Moghbel et al. 2015; Istiqomah and Shofi 2018; Pan-pan et al. 2018). The high concentration of colchicine may be lethal to sensitive plant tissues (Pan-pan et al. 2018). Increasing ploidy is an efficient way to create superior plants to sterile plants such as garlic by improving the morphology, disease resistance, adaptability to environmental stress, and yield or quality. Chromosome doubling agent and type of in vitro explants are the major factors that affect the induction of plant polyploidy. The chromosome doubling agent should be efficient with low toxicity, and the explant should be reliable in propagation (Cheng et al. 2012). The variation in ploidy level within the same treatment condition could be due to the stage of the cells that are responsible for the initiation of new shoots. A number of cells in meristematic tissues exhibited polyploidy (Gantait et al. 2011). Lower survival rates of explants are prevalent in high concentrations of colchicine and longer treatment time.

The main component in raw garlic is alliin. When the clove of garlic is crushed or cut, the cell walls are split so that an enzyme called alliinase is released, which converts alliin into allicin. Allicin is the chemical that gives garlic its odor. It creates free radicals in human body, which in excess, can be dangerous. Allicin can cause stomach irritation and, in rare cases, hemolytic anemia, destruction of red blood cells. If placed directly on human skin, allicin can cause blistering. During cooking, allicin produces ajoene, DADS (diallyl disulfide), and other compounds that may help keep blood from clotting (Coleman 2002).

Conversion of (＋)-alliin to allicin in garlic via the actions of allinase and pyridoxal phosphate.

Determining ploidy level as an artificial induction is an important resource for breeding programs for the production of new cultivars. Increasing ploidy level is affected by different plant materials, time exposure, and concentration of the anti-mitotic agent (Yu et al. 2013). But it also has some lethality in the process of chromosome doubling of explant. The interaction between colchicine concentration and treatment time, type of explant, and other factors would affect the survival rate of explants induction. High concentration and longer duration of colchicine could reduce the survival rate, whereas low concentration with longer duration of colchicine resulted in a higher polyploidy induction rate (Yu et al. 2013; Pan-pan et al. 2018).

Colchicine affects chromosome doubling on mitotic cells by binding to the microtublin positive end leading to the disruption of the polymerization of microtublin (Moghbel et al. 2015; Zhang et al. 2016; Roughani et al. 2017). The spindle fibers consisting of microtubules used to pull the sister chromatids to opposite poles of the cell. However, without the action of spindle fibers, the mitotic process is disrupted. This disruption results in DNA replication without cell division (Roughani et al. 2017). Wei et al. (2007) reported the increased number of chromosomes brings the change in plant morphology and functions. The new seedlings have vigorous mitosis cells. When the seedlings grow up, colchicine will lose its function on double chromosomes. Usually, seedlings grow very slowly after treatment, because residual colchicine will be left and harm the newly tender buds. Common methods for chromosome doubling involve soaking the seeds in a colchicine solution, applying colchicine using a brush on growing shoot apices, or culturing (in vitro) plantlets in a colchicine-containing medium (Spencer-Lopes et al. 2018). Manipulation of the ploidy level is an important tool in improving crops. The use of chemicals to induce changes in chromosome number by blocking at metaphase of mitosis. Colchicine bind to the tubulin dimmers and inhibiting the formation of microtubules and spindle fibers during cell division. Colchicine is toxic to plants, hence, low concentration reduces its toxic effects and enhance the production of polyploids (Sajjad et al. 2013). The duration of colchicine treatment is a critical factor for chromosome doubling. The duration of colchicine treatment has a significant effect on chromosome doubling induction and survival rate (Mohammadi et al. 2012; Roughani et al. 2017).

Colchicine inhibits microtubule polymerization by binding to tubulin, and inhibits the development of spindles or destroys those already present, hence the cell cannot move its chromosomes around and the cell may end up copying the chromosomes and cannot parcel them out into new cells and so it never divides (Koyani and Saiyad 2011; Sajjad et al. 2013). Colchicine prevents spindle formation at prophase, impedes nuclear mitosis, delays chromosomal separation, inhibits daughter nuclei, and efficiently blocks the cleavage process. Hence, when root tips or other growing plant parts are placed in appropriate concentration of colchicine, the chromosomes of treated cells duplicate without spindle formation and the cytoplasmic phase of cell division would not occur (Lindayani et al. 2010).

Garlic treatment by colchicine in vitro

A schematic diagram of workflow mutation using colchicine and use of in vitro techniques in garlic.

Polyploidization plays a key role in plant breeding and crop improvement. Polyploid plants possess three or more sets of homologous chromosomes. The increase in chromosome number in these plants is the result of a genome duplication event (Corneillie et al. 2019). Polyploidy has been induced by colchicine in several crops (Datta 1990). Polyploidy (whole-genome duplication) occurs naturally due to the formation of unreduced gametes or artificially induced by doubling the number of chromosomes in somatic cells (Manzoor et al. 2019). Polyploid plants have a larger stoma and epidermis size but fewer stomata density than control plants (Setyowati et al. 2013). Polyploid plants have lower stomatal density compared to diploid plants due to the enlarged size of stomata per leaf area (Table 3) (Ayu et al. 2019).

Table 3 . Morphological and chemical traits of diploid and polyploid garlic plants.

Colchicine-treated plants	Leaf area index	Stomatal length (mm)	Stomatal width (mm)	Stomatal area (mm2)	Total soluble sugars (g g–1 FW)
Diploid (Control) (2n = 16)	3.01 ± 0.05b	28.20 ± 0.20b	20.90 ± 0.11b	589.28 ± 1.89b	0.33 ± 0.01b
Tetraploid (4n = 32)	9.02 ± 0.49a	56.86 ± 0.26a	33.16 ± 0.09a	1,884.65 ± 9.388a	0.48 ± 0.05a
LSD (P ≤ 0.05)	1.61	5.56	3.23	20.4	0.03

Adapted from Dixit and Chaudhary (2014): The Leaf area index is calculated using a leaf area meter. Total soluble sugar concentrations (TSSC) are measured in fully-mature garlic bulbs.

Colchicine is a chemical mutagen used for polyploid induction in vitro. Polyploid plants retain long stomata (Münzbergová 2017), larger and thicker leaves (Jaskani et al. 2005; Sajjad et al. 2013; Moghbel et al. 2015; Roughani et al. 2017), high chloroplast number (Jaskani et al. 2005), lower number of stomata compared to control (Sajjad et al. 2013), higher secondary metabolites (Tavan et al. 2015) and increased sugar composition (Dixit and Chaudhary 2014; Corneillie et al. 2019) used in overcoming hybridization barriers, improving stress tolerance, and restoring fertility in wide hybrids (Roughani et al. 2017). Polyploid seeds are often larger than diploid seeds because of their large cell size in polyploids (Moghbel et al. 2015; Tavan et al. 2015; Münzbergová 2017). Moghbel et al. (2015) described polyploids induced by colchicine in vitro results in an increase in DNA content in plants and affects the epidermal surface cells. Polyploids have a thicker cell size but have a low cell number per leaf blade as compared to the diploids (Corneillie et al. 2019). Colchicine treated plants have larger stomata, morphological features such as shoot and leaf thickness (Moghbel et al. 2015; Tavan et al. 2015). Root length is the result of cellular extension in the root system. Specific colchicine concentration and time duration decrease root and shoot length, due to the disruption of cell division that hinders plant growth (Sajjad et al. 2013; Ayu et al. 2019). Plant of Zingiber officinale Rosc. treated with colchicine does not exhibit any significant difference in root length compared to the control (Ariyanto 2011). In Gerbera jamesonii Bolus cv. Sciella, 0.1% colchicine treatment increases root length significantly than the control (Gantait et al. 2011). High colchicine concentration has a negative effect on root formation and cell division rate (Heo et al. 2016) and shoot proliferation (Sajjad et al. 2013). High colchicine concentration leads to a death of explants due to much stress on plant cells.

Polyploid plants possess a higher leaf areal index (Pharmawati and Waitiani 2013), length and width of stomatal guard cells (Münzbergová 2017), and total soluble sugar contents (TSSC) (Corneillie et al. 2019) than in control diploid garlic plants (Table 3). The mean of morphological traits of colchicine treated plants increases significantly with an increase in ploidy level. However, polyploidy plants have a smaller length:width ratio to diploid control plants (Dixit and Chaudhary 2014).

Chimera

The main problems in mutation breeding of vegetatively propagated plants in vitro are the formation of chimeras and the somatic elimination (diplontic selection) of mutated sectors after mutagenic treatment (Bahadur et al. 2015). These phenomena can be overcome by axillary and/or adventitious bud differentiation from single cells during in vitro culture. This morphogenetic pathway of plant regeneration can avoid chimeras, and increase the mutant frequency, and probably broadens the mutation spectrum. Results in garlic are promising and research on in vitro mutation breeding technology has been established (Broertjes 2012). All cells exposed to mutagen do not incur mutations, but those that do incur mutations will give rise to cells reveal the mutation (Bahadur et al. 2015).

Chromosome counting

Polyploidization determination classified into direct (chromosome counting in mitotic cells of root tips) and indirect (cytologic characteristics such as the size of stomata cells, stomata density, and number of chloroplasts in guard cells). Chromosome counting is the most intuitive and reliable method for the identification of the level of ploidy. Ploidy identification can be performed through chromosome counting, flow cytometry analysis, external morphology observation, and stomatal guard cell observation (Li et al. 2019). However, each method has its disadvantages. Chromosome counting is an accurate method but it is time-consuming and difficult to conduct; flow cytometry analysis is fast with high fluctuation, but it is more relatively accurate; whereas, external morphology and stomatal guard cell observation are simple but not accurate enough (Li et al. 2019). Chromosome counting should be performed at the mitotic metaphase stage when the chromosomes become fully contracted (Fig. 1). During the process of chromosome preparation, the selection of pretreatment chemical solution and treatment time are the key parameters that could affect the shape of images on metaphase mitotic (Jaskani et al. 2005; Pan-pan et al. 2018). Chromosome counting is determined by flow cytometry and morphological analysis. Chromosome counting in the mitotic cell of root tips determine the ploidy level, but it is time-consuming and requires much experience. Nevertheless, chromosome counting will still be important to ensure polyploidy production from the colchicine treatment (Yenchon 2014).

Figure 1. DNA content and cell cycle.

Colchicine is effective to induce ploidy in garlic at a concentration of 0.001%-1% in a period of 6-72 hours immersion, depending on the response of each plant species (Amanah et al. 2016). According to Dixit and Chaudhary (2014), Ploidy induction of diploid garlic genome can be induced by treating garlic stem discs up to 0.75% colchicine with higher duplication at 0.5% and lower duplication rate at 0.75% due to higher toxicity (Table 4). The application of colchicine helps to increase the ploidy level and an increase in ploidy is expected to make the bulb size larger in garlic. Larger tuber size can increase the total tuber weight and also the overall garlic productivity.

Table 4 . Effect of different concentrations of colchicine on morphological charactersitics and chromosome counts in garlic (Allium sativum).

Morphological character	Isolate/clone	Chromosome count (2n)	Size of stomata (relative to diploid control)*	Colchicine concentration (w/v)
Dark-green leaves (DG)	DG 1	16	S	0.25
	DG 2	16	D	0.25
	DG 3	16	S	0.25
	DG 4	24	D	0.25
	DG 5	16	S	0.25
	DG 6	16	S	0.25
	DG 7	18	D	0.50
	DG 8	16	S	0.50
	DG 9	Multiple sets	I	0.50
	DG 10	Multiple sets	I	0.50
	DG 11	16	S	0.50
	DG 12	16	S	0.50
	DG 13	16	S	0.50
	DG 14	Multiple sets	D	0.50
	DG 15	Multiple sets	I	0.50
	DG 16	16	S	0.75
	DG 17	16	S	0.75
	DG 18	16	S	0.75
	DG 19	Multiple sets	I	0.75
	DG 20	16	S	0.75
	DG 21	16	S	0.75
Reduced growth (RG)	RG 1	8	D	0.25
	RG 2	8	D	0.25
Green and thick leaves (GT)	GT 1	32	I	0.50
	GT 2	32	I	0.50
	GT 3	32	I	0.50
	GT 4	32	I	0.50
	GT 5	32	I	0.50
	GT 6	32	I	0.50
	GT 7	32	I	0.50
	GT 8	32	I	0.50
	GT 9	32	I	0.50
Curled leaves (CL)	CL 1	Multiple sets	D	0.75
	CL 2	Multiple sets	D	0.75
	CL 3	Multiple sets	D	0.75
	CL 4	Multiple sets	D	0.75

*Adapted from Dixit and Chaudhary (2014): Stomatal measurements were made on the lower epidermis of the second fully-expanded leaf. S: Similar sized stomata to control diploid plant. D: Decreased stomatal size compared to diploid control. I: Increased stomatal size compared to diploid control.

Cytological analysis provides information for polyploidy identification. The quick, early, and clear-cut detection of polyploid plants from colchicine-treated plant populations is a fundamental requirement for genetic improvement programs based on chromosome duplication (Sajjad et al. 2013). The overall size of the nucleus and chromosome number, plantlets regenerated from colchicine-treated garlic explants have enlarged nuclei as compared to normal plants (Sinha et al. 2016). Cytogenetic studies are crucial for getting information about the role and effects of mutagens and revealing the responses of different crop genotypes to a particular mutagen (Udensi and Ontui 2013). The ploidy status/DNA content of the regenerated plants is determined using flow cytometry (Mohammadi et al. 2012). To determine the basic somatic ploidy level, a small leaf fragment (1 cm²) from each plant should be harvested and chopped using a sharp razor blade (Corneillie et al. 2019). Flow cytometry measures DNA quantity in nuclei from fresh leaves. Ploidy level analysis is laborious and time-consuming. Although it is a quick and hardly destructive technique, it measures nuclei in phases of high and low ploidy levels (Foshi et al. 2013; Yenchon 2014). Roughani et al. (2017) reported flow cytometry is a powerful technique with a fast and accurate method for the estimation of DNA content, has become the principal technique for establishing plant genome size. Jaskani et al. (2005) reported as flow cytometry is used for ploidy analysis, and it is the most accurate tool for ploidy determination. It is a non-destructive tool that requires a small amount of tissue to analyze large populations of cells where mixoploidy or aneuploidy exists. Tetraploid garlic plants show slow growth and are characterized by thick dark-green leaves compared to diploid plants. The shortest peak in the DNA histogram represents the total nuclei in G₁ and G₂ phases (Fig. 2). Mechanical chopping during cell nuclei extraction does not produce significant DNA hydrolysis or nuclear degradation (Galbraith et al. 1983). Many plant tissues do not yield enough protoplast; hence it is difficult to confirm the extracted protoplast can represent the entire plant population. Mechanical chopping for cell nuclei extraction may result in biases and random selection of plant tissue nuclei to be analyzed (Galbraith et al. 1983). There are different fluorochromes for staining the extracted double stranded DNA with different wavelength excitation and emission capacities (Table 5).

Table 5 . Common fluorescent dyes for nuclear DNA staining.

Fluorochrome	Primary binding mode	Wavelength (nm)*
		Excitation	Emission
Ethidium bromide**	Intercalation	530	605
Propidium iodide**	Intercalation	540	615
Hoechst 33258	AT-binding	365	465
Hoechst 33342	AT-binding	360	460
DAPI (4,6-diamidino-2-phenylindole dihydrochloride)	AT-binding	365	450
DIPI (4’,6-bis (2’-imidazolinyl-4H.5H)-2-phenylindole)	AT-binding	365	450
Chromomycin A3	GC-binding	445	570
Mithramycin	GC-binding	445	575
Olivomycin	GC-binding	440	560

*Dye-DNA complex. **Binds also to double-stranded RNA.

Figure 2. Analysis of nuclear DNA content. Adapted from Doležel and Bartoš (2005), Doležel et al. (1994), and Tuwo and Indrianto (2016).

Callus production, cell proliferation, shoot proliferation from callus, and regeneration can be affected by the composition of the culture medium, the combination of growth regulators used, and the kind of explants. Garlic responds well to Murashige and Skoog (MS) medium, sometimes half-strength (Murashige and Skoog 1962), and Basal Dunstan Short (BDS) medium. Some studies also used Gamborg’s B5 medium for garlic micropropagation. Sinha et al. (2016) described explants cultured on Murashige and Skoog (MS) medium supplemented with BAP (Benzylaminopurine) and Kinetin at various concentrations ranging from 1.0-5.0 mg/L either alone or in combination are suitable for garlic proliferation. According to many studies in garlic, an optimal concentration of Kinetin and BAP (Benzylaminopurine) is also used for inducing polyploidization. The use of meristems as initial explants and 2,4-D (2,4-dichlorophenoxyacetic acid) as the appropriate growth regulator can enhance callus induction. Garlic induction at low and high 2,4-D level is not effective in callus induction and organogenesis. Callus induction is better in both 2,4-D (2,4-dichlorophenoxyacetic acid) and kinetin combinations. However, both growth hormones are not effective in shoot proliferation and regeneration. Auxins and cytokinins in combination are effective in producing callus instead of auxins alone in garlic. Callus proliferation is reported better in Basal Dunstan Short (BDS) medium supplemented with 1.5 mg/L 2,4-D ＋ 1.0 mg/L kinetin; and Murashige and Skoog (MS) medium supplemented with 1.5 mg/L 2,4-D ＋ 1.0 mg/L kinetin (Hailu et al. 2020). The development of a somatic embryo into a whole plant can be affected by the non-optimal growth hormone composition during the callus growth. The developmental processes, such as callusing and proliferation are controlled by plant growth regulators. Plant growth regulators concentration is vital for an optimum response as too low concentration may not trigger callus induction, cell proliferation, and shoot proliferation. The combination of Kinetin and 2,4-D (2,4-dichlorophenoxyacetic acid) causes slightly better responses in callus induction than the use of 2,4-D (2,4-dichlorophenoxyacetic acid) alone in most garlic improvement programs by mutation breeding in vitro.

In vitro plants decrease their survival rate when the concentration of colchicine and in vitro culture increases with time. Colchicine treated explants can survive even at higher colchicine concentrations in vitro, but there is also a higher mortality rate of explants due to toxicity (Table 4). Amanah et al. (2016) reported that colchicine is effective in inducing diploid plants at a concentration of 0.001-1% in a period of 6-72 hours depending on the response of each plant species. High colchicine concentration leads to higher plant death as compared to the untreated plants. LC50 (lethal concentration 50%) is used to determine the optimum concentration to be used in the mutagenesis experiment and it causes 50% lethality in the plant organism used for irradiation in defined time duration. LC 50% varies with the plant species, the type and status of the material, and the stage where the lethality is measured. The colchicine treated explants usually have better morphology compared to the untreated explants of garlic (Table 3). The use of a high concentration of colchicine in vitro may have several negative effects on the physiological process of treated plants due to the disruption of the genetic content of the plants (Ayu et al. 2019). Polyploid plants possess large and thicker leaves and shoots with a low number of stomata and high chloroplast numbers compared to the diploid plants (Münzbergová 2017; Roughani et al. 2017; Ayu et al. 2019). Tuwo and Indrianto (2016) reported that plantlets at a lower colchicine level grew faster than plantlets that have higher colchicine levels. The degree of genetic variability obtained from in vitro culture depends on the genotype, type of explant, growth hormone, and the culture conditions used (Mubarrat et al. 2018). Colchicine treated explants are not effective in producing roots in vitro. Limiting the growth and development of roots may affect the plant’s ability to absorb nutrients from the growth media due to cell division failure. Cytokinins inhibit rooting and affect colchicine treatment efficiency in the growth medium. Many economically important plant species cannot induce root easily. The reluctance to rooting could be due to a high level of cytokinin persisting in stem tissues. However, rooting can be induced by the transfer of the shoots once or twice to medium without cytokinin. Plant genotype has an adverse effect in shoot and root growth and differentiation, and adventitious root induction during plant tissue culture.

Flow cytometry measures DNA quantity in nuclei from fresh leaves. Ploidy level analysis is laborious and time-consuming. Although it is a quick and hardly destructive technique, it measures nuclei in phases of high and low ploidy levels. 0.5 cm² of fresh leaf from the middle part of the leaf can be used to extract cell nuclei during flow cytometry analysis. The extraction can be conducted manually by grinding the plant tissues through a laser blade in a petri dish. Staining can be performed with Propidium iodide (PI), DAPI (4,6-diamidino-2-phenylindole dihydrochloride), or DIPI (4’,6-bis (2’-imidazolinyl-4H.5H)-2-phenylindole), and Mithramycin or Olivomycin. Propidium iodide (PI) is a red fluorescent stain that preferentially intercalates the double-stranded DNA with 540 nm wavelength excitation and 615 nm emission; while, both DAPI and DIPI binds A-T nucleotides within the double-stranded DNA with 365 nm wavelength excitation and 450 nm emission. And Mithramycin binds G-C nucleotides within the DNA double-strand with 445 nm excitation and 575 emissions; while Olivomycin binds G-C nucleotides with 440 nm excitation and 560 emissions (Table 5) (Galbraith et al. 1983; Otto and Tsou 1985).

Colchicine causes microtubule depolymerization and inhibits cell division by blocking microtubule development. Microtubule depolymerization causes the disturbance of the spindle thread formation during cell division. The disturbance of spindle fibers results in the separation of chromosomes but the cells fail to produce new cells and cause the doubling of chromosomes. During colchicine treatment of explants, some cells may not be affected by colchicine treatment and remain as diploid (2n). Usually, only partial induction of chromosome cells remains doubled by colchicine treatment (Amanah et al. 2016). The application of colchicine treatment in vitro can increase the ploidy level in garlic, and an increase in ploidy is expected to have a larger bulb size garlic varieties. Larger tuber size is expected to increase the overall tuber weight and garlic production.

Meristem-tip culture is an effective method of pathogen eradication in vegetatively propagated garlic species. Production of virus-free garlic in tissue culture should be established to produce healthy plants because garlic remains susceptible to virus reinfection in field conditions. Meristem-tip cultures are valuable for garlic germplasm conservation. Meristem-tip explant cultures are suitable systems for mutation by colchicine and polyploidy induction in garlic. Colchicine-treated garlic meristem culture can generate new genetic variability via colchicine treatment which can be useful for the improvement of garlic crops. The degree of genetic variability obtained from in vitro induction depends on genotype, type of explant, growth hormone, and the culture conditions used. Lower auxin and cytokinin concentrations are effective hormones for callus production and cell proliferation in garlic. 2,4-D (2,4-dichlorophenoxyacetic acid), and Kinetin or 2,4-D (2,4-dichlorophenoxyacetic acid) and BAP (Benzylaminopurine) in Basal Dunstan Short (BDS) or Murashige and Skoog (MS) medium are key hormones in callus induction and cell proliferation. However, different garlic genotypes have different responses to growth mediums and hormones. Further research is needed to identify the optimal sources of explants, sterilization process, the concentration of colchicine in vitro, time duration for garlic polyploidy induction as well as the protocols for shoot proliferation and regeneration in garlic. In many studies, Murashige and Skoog (MS) medium supplemented with different concentrations of BAP (Benzylaminopurine) and Kinetin showed a wide variation in a shoot-root initiation and proliferation of garlic varieties. Auxins are used to induce callus, cell proliferation, and somatic embryogenesis while cytokinins induce shoot and root differentiation and elongation. The use of meristematic explants and 2,4-D (2,4-dichlorophenoxyacetic acid) as a growth regulator enhances callus induction. The addition of cytokinin at 1.0 mg/L along with 1.5 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) can increase callus induction and cell proliferation. Callus differentiation and plant development are determined by growth regulators. An efficient system for fast production of callus and subsequent regeneration of garlic is a problem in in-vitro culture.

The genetic diversity of local garlic varieties can be enhanced by colchicine treatment and immersion time. At lower level colchicine treatment, there is a possibility of producing higher mixoploid plants as compared to higher colchicine treatment. While, at higher colchicine treatment, high ploidy induction and higher mortality is prevalent. Tetraploids possess thicker and darker green leaves with slow growth compared to the untreated diploid plants. The application of colchicine at 0.25-0.5% and immersion time at 24, 36, and 48 hours can improve the genetic potential of local garlic varieties in vitro.

Polyploidization plays a key role in plant breeding and crop improvement. Polyploid garlic plants possess lower stomatal density, thicker leaves, higher chloroplast number, higher secondary metabolites, and slower root and shoot formation compared to diploid plants. Polyploids have a thicker cell size but have a low cell number per leaf blade as compared to the diploids. In vitro culture with colchicine treatment enhances the frequency and the spectrum of chromosomal mutations. Genetic diversity caused by colchicine treatment is used as a source of germplasm for plant breeding programs. The genetic diversity in garlic as a result of colchicine treatment aids to know the different characters of genes, so that it can be used for the finding of superior cultivars in garlic varieties.

The authors declare that they have no conflicts of interest.