Polyploidization is a tool used either under in vivo and
Generally, mitotic polyploidization is based on the application of antimitotic agents. Colchicine, the widely using chromosome doubling agent, amiprophos-methyl (APM), butamifos, oryzalin, propyzamide, trifluralin have been successfully tested for different species (Hansen & Andersen 1996, 1998; Petersen et al. 2003; Cheng et al. 2012; Orcen & Emiroglu 2014). Nowadays, the substitution of antimitotic agents with compounds of lower toxicity to humans than colchicine, is widely testing across different crops. It has been reported that trifluralin, the component of herbicides, is more specific for plant tubulin and mitosis
Cabbage (
Seeds of six genotypes of white head cabbage, cultivars ‘Flexima’ (F), ‘Mutsuma’ (M), ‘Septima’ (S), ‘Zeus’ (Zeu), ‘Ancoma’ (Anco), and breeding line DC6 obtained from MoravoSeed CZ a.s. (Czech Republic) and Reprosam s.r.o. (Czech Republic) were used in this study. Three different explants, seeds, hypocotyl, and organogenic calli were used. Hypocotyl segments were excised from 6-day old
Explants were subjected to various trifluralin concentrations (0.5, 1.0, 1.5, and 2.0 mg·L−1) and treatment conditions (24 °C/24 h, 24 °C/48 h, 30 °C/24 h, and 30 °C/48 h in dark). Hypocotyl and calli explants were incubated in liquid MS medium supplemented with vitamins as above, sucrose (20 g·L−1), and trifluralin (each concentration separately) at pH 5.8. Seeds were subjected to the same trifluralin treatments in half-strength MS medium. The control treatment was conducted using the same liquid MS medium without trifluralin. Two replicate trials were conducted for each treatment. Each trial involved four Petri plates with six calli, ten hypocotyls, and seven seeds. After the successive treatment, explants calli, seeds, and hypocotyls were washed three times and cultured on MS callus induction medium, MS seed germination medium mentioned above and MS shoot induction medium supplemented with vitamins as above, plus NAA (0.2 mg·L−1), BAP (3 mg·L−1), GA3 (0.01 mg·L−1), AgNO3 (0.5 mg·L−1), sucrose (20 g·L−1), and phytagel (7 g·L−1) at pH 5.8. After one week, calli were transferred to fresh same MS shoot induction medium. In every two weeks, shoots produced on calli and hypocotyls were repeatedly transferred to a fresh medium of the same composition and incubated at the culture conditions described above until developing typical and possible to dissect from mother explant small shoots (1–2 cm height).
Shoot tips of seven days old seedlings derived from treated seeds were excised and cultured on MS shoot development medium supplemented with vitamins, NAA (0.2 mg·L−1), BAP (3 mg·L−1), GA3 (0.01 mg·L−1), sucrose (20 g·L−1), and phytagel (7 g·L−1) at pH 5.8. Cultures were incubated for several weeks with repeated transferring into fresh media till they reached 2–3 cm height. MS rooting medium supplemented with IBA (0.2 mg·L−1), sucrose (10 g·L−1), and phytagel (7 g·L−1) at pH 5.8 was used to induce roots, and incubated at 25 ± 2 °C in light for maximum 8 weeks.
Following trifluralin treatment, the number of seeds germinated after 7 days on germination medium, the number of shoots derived per germinated seed, and the number of shoots with induced roots were recorded to analyze the effect of trifluralin on
The ploidy level of each successfully grown plant was analyzed by flow cytometry using leaf tissues from successfully developed plantlets. About 1 cm2 leaf tissue was chopped with a razor blade in 0.5 ml of Otto I buffer [0.1 M citric acid monohydrate and 0.5% (v/v) Tween 20] and the suspension was filtered through a 42 μm nylon mesh. The filtrate was incubated for 10 min at room temperature and 1 ml of Otto II buffer (0.4 M Na2HPO4·12 H2O), 4 μg·mL−1 4,6-diamidino-2-phenylindole, and 2 μL·mL−1 β-mercaptoethanol were added. The fluorescence intensity was measured using a CyFlow Space flow cytometer (Partec GmbH, Münster, Germany).
All the results were analyzed using factorial analysis of variance (Factorial ANOVA) with statistical software, Statistica version 13.3, and significant differences were assessed by least significant difference (LSD) at p = 0.05. The main effect of each factor, cultivar, trifluralin concentration, and treatment condition, rate of seed germination, rate of shoot induction, and rate of root induction individually, and the interaction effect between each factor were tested.
Nowadays, lack of proper knowledge of
Among the three types of explants tested in this study, hypocotyl and calli showed a poor response for
A similar type of response was observed from the treated calli as well. Most calli turned brown and died within one to two weeks on callus induction medium. Calli that showed growth after the treatment, either became green and dormant with zero shoot induction (Fig. 1) or shoot primordia induced from calli did not elongate. None of calli and hypocotyls of any cultivar tested resulted in successfully developed plantlets. It seemed that the tested concentrations and conditions were harsh and lethal for those fragile and sensitive explants. Cheng et al. (2012) used garlic calli to induced polyploids using trifluralin and achieved tetraploids but they also observed a reduction of survival rate of calli up to 20% when the concentration increased to 200 mM (67.06 × 103 mg·L−1) with 15 days incubation period. Furthermore, they observed a reduction of shoot differentiation rate by 26.7% with the increase of treatment strength.
Compared to hypocotyl and calli, seeds were hopeful explants for the study. Seed germination was varied among cultivars. Compared to control, most cultivars showed a significant decrease in germination rate after the trifluralin treatments (Table 1). Germination was varied from zero to 100% depending on the treatment. For example, cultivar ‘Mutsuma’ did not germinate at 1.00 mg·L−1, 24 °C/24 h, and 30 °C/24 h, but the cultivar ‘Septima’ showed 100% germination at 0.5 mg·L−1, 24 °C/24 h, and 1.00 mg·L−1, 24 °C/48 h. The variation in germination rate after treatments was significantly different between cultivars, treatment conditions, and concentrations tested (Fig. 2). Furthermore, a significant interaction between cultivar, treatment condition, and trifluralin concentration (Table 1) was noticed. With increased temperature, incubation period, and trifluralin concentration, the seed germination rate declined in all cultivars.
Results of ANOVA to test the influence of cultivar, trifluralin concentration, treatment condition on
Trait | Effect | MS | F | P |
---|---|---|---|---|
Rate of seed germination | cultivar | 11339.3 | 87.4 | 0.0000* |
trifluralin concentration | 33654.1 | 259.3 | 0.0000* | |
treatment condition | 7613.4 | 58.668 | 0.0000* | |
treatment condition × cultivar | 638.6 | 4.9213 | 0.0000* | |
treatment condition × concentration | 382.4 | 2.9465 | 0.0015* | |
concentration × cultivar | 925.4 | 7.131 | 0.000* | |
treatment × concentration × cultivar | 237.4 | 1.829 | 0.00262* | |
Shoots per germinated seed | cultivar | 17.91 | 2.813 | 0.014* |
trifluralin concentration | 4.4 | 0.692 | 0.5990 | |
treatment condition | 6.5 | 1.021 | 0.3867 | |
treatment condition × cultivar | 10.05 | 1.5795 | 0.0799 | |
treatment condition × concentration | 9.34 | 1.4674 | 0.1492 | |
concentration × cultivar | 9.53 | 1.4972 | 0.0860 | |
treatment × concentration × cultivar | 6.96 | 1.094 | 0.337 | |
Rate of root induction | cultivar | 10575.3 | 47.1 | 0.0000* |
trifluralin concentration | 23284.8 | 103.6 | 0.0000* | |
treatment condition | 6557.2 | 29.186 | 0.0000* | |
treatment condition × cultivar | 377.3 | 1.6793 | 0.05559 | |
treatment condition × concentration | 717.0 | 3.1912 | 0.00068* | |
concentration × cultivar | 1636.5 | 7.2842 | 0.0000* | |
treatment × concentration × cultivar | 446.7 | 1.988 | 0.000749* |
The seven-day-old seedlings were transferred into MS shoot induction medium for shoot multiplication and subcultured three times into fresh same medium within 3–4 weeks. The number of shoots obtained from one germinated seed significantly varied between cultivars (Table 1) and was not different from control. The effect of incubation temperature, duration, and trifluralin concentration on shoot multiplication was not statistically significant (Fig. 2).
The effect of trifluralin concentration, incubation temperature, and duration of root induction was significant as well as the differences between cultivars were also significant (Fig. 2). Even though the effect of treatment conditions and cultivars itself on root induction was highly significant (Table 1), no significant interactions between them (Table 1) were observed. According to Meyer et al. (2009) and Allum et al. (2007), the exposure time and concentration of the antimitotic compound and the type of explant, significantly affect survival rate and ploidy induction of
The success of ploidy induction depends on the permeability of the explant tissues and penetration of the particular antimitotic agent together with the exposure time, concentration and temperature (Allum et al. 2007). The treatment conditions and concentrations used in this study could be harsh on the viability of the majority of seeds but was not sufficient to induce polyploids from viable seeds due to poor cell permeability to have proper translocation of the chemical for its function as an anti-tubulin agent resulting in zero polyploids.
The ploidy level of
We observed a significant reduction of seed germination with the increase of trifluralin concentration and incubation temperature and duration. The shoot multiplication increased with increased incubation temperature and duration though it reduced with increased concentration. Thus, in the future, we plan to conduct experiments on the effect of increased temperature, incubation period, and a lower concentration of trifluralin aiming to increase the efficiency of polyploid induction. Furthermore, it is possible that the use of pre-induced germinating seeds to increase chemical penetration would be successful in achieving induction of polyploids in cabbage.
From three different types of explants tested, seeds can be most prospective as the potential explants for further studies on establishing of