Polyploid plants (polysomic polyploids, true, and segmental amphidiploids) can be produced using various chemicals, such as colchicine, amiprophos-methyl, trifluralin or oryzalin; or physical methods such as temperature shock and protoplast fusion [8]. The most common chemical is colchicine obtained from Colchicum autumnale L., which has an inhibitory effect on the spindle formation, causing a failure during anaphase disjunction [8] and cytokinesis [7], thus resulting in one cell with the doubled chromosome set. Hence, colchicine treatment results in either pure polyploid individuals or impure (mixoploid or aneuploid cells), depending upon the colchicine concentration and exposure time, plant tissues and its development stage, and method for chromosome set doubling. The colchicine solution may be applied to immature buds, resulting in unreduced reproductive cells and, subsequently, producing polyploidy seed at different rates. It is applied to the pedicel of growing immature bud or the inflorescence stalk may be dipped in a growth media solution with low concentration of colchicine (0.01 to 0.1%), depending upon the forage species (Table 1). Cotton swab dipped in colchicine solution of 0.2% for 10 h during 2 consecutive days successfully induced a polysomic tetraploid (12–40%) in guar accessions [15]. Colchicine can also be applied to the callus; i.e., cell culture of meristematic tissues to produce chimeric plants, or seed may be germinated in a colchicine solution [16]. Generally, colchicine (0.1‒1%) has been applied to seeds for 24–48 h depending upon the forage species to target cells undergoing mitosis (Table 1). Growth arrest is the first symptoms of successful application of colchicine (Figure 1). Affected cells usually resume division after a lag period of colchicine application. A comparison of euploid series (2×, 4×, 6× and 8×) in thale cress (Arabidopsis thaliana L. Heynh.) showed that induced polyploidy had slower growth, enlarged cell size, and decreased the number of cells per leaf blade. Polyploid cells had lower lignin and cellulose but higher pectin and hemicelluloses in the stem [17]. Frequency of polyploidy induction can increase by rising colchicine concentration (Table 1). A high colchicine concentration has been, however, toxic to cell and plant tissues. A low colchicine concentration (0.1%) treatment with relative long duration (48 h) has been effective in increasing the yield of polyploid cells in shoot tips in vitro [18] (Table 1). For example, sorghum (Sorghum bicolor (L.) Moench.) seed were subjected to 0.2% concentration of colchicine for 48 h and 72 h, yielding 3.3% and 2.3% polysomic tetraploid plants, respectively [19]. A direct application of colchicine by dipping the apical meristem of seedlings produced chimeric tissues where induced polyploidy was limited to a particular layer [20].

 

Polyploid plants can ensue from somatic cell fusion [21]. This method has been used to fuse protoplast of the same species (polysomic polyploidy) or from two species (amphiploidy or disomic polyploidy) in an electric shock chamber without reduction division, thus obtaining amphidiploid forage species [22].

 

In vitro induction of polyploidy was successfully achieved by culturing the nodal segments or callus in tissue culture media supplemented with a low concentration of colchicine. This method induces high polyploidy frequency in various species (Table 1). Although other chromosome doubling agent such as, oryzalin had a higher frequency of induced polyploidy than colchicine [23], it inhibited callus growth and seedling regeneration. Liquid medium was more effective in inducing polyploidy than semi solid media [7,23]. A concentration of 5 µm oryzalin was more effective than colchicine and trifluralin in inducing chromosome doubling of calli obtained from interspecific hybrid plants of elephant grass × pearl millet [24]. In vitro treatment of red clover (Trifolium pratense L.) yielded 55.5% more polysomic tetraploids and 1.9 times fewer chimeric individuals than imbibition of seed in a colchicine solution [25,26]. Colchicine, amiprophosmethyl and oryzalin produced similar frequency of polysomic tetraploid (31–47%) and chimeric plants (14–22%) in T. pratense [25]. Colchicine (0.015% in tissue culture media) was more effective than oryzalin in inducing polysomic tetraploidy (16%) in peanut clover (Trifolium polymorphum Poir.) [27].

 

The demand for pure polyploids with different ploidy levels obtained after chromosome doubling needs screening, selection and periodic evaluation of the ploidy level stability of the natural and induced [28]. Nowadays, the main method used for this purpose is flow cytometry [8,29], which increases the efficiency of ploidy level determination and nuclear genome size measurement. With flow cytometry large numbers of individuals are quickly evaluated in only one day using a quantitative, rapid, reliable and reproducible method [11]. This powerful screening tool for ploidy level has been very useful in  breeding programs. However, chromosome counting, which is performed through microscopic observation of the metaphase cells, has been applied to confirm the chromosome number [8,28,29], and to detect eventual aneuploids. Besides these direct tools, indirect analyses have also been accomplished. The effects of induced polyploidy on forage species were noted as an increase in the stomatal area and a decrease in the stomatal frequency. These changes in leaf anatomy arose after expanding cell volume due to increasing nuclear DNA content [30].