The Effect of Trifluralin on Post-in vitro Morphogenesis of Five Genotypes of Head Cabbage (Brassica oleraceae var. capitata)

Polyploidization is a tool used either under in vivo and in vitro conditions in crop breeding programs to obtain a good source of material for further selection and/or hybridization. Mitotic and meiotic polyploidization, are the two different ways employed in artificial polyploids induction.

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 in vitro with less phytotoxicity and efficient chromosome doubling (Khosravi et al. 2008).

Cabbage (B. oleracea var. capitata) is a well-known diploid vegetable containing high amounts of antioxidants such as glucosinolates. There are many reported studies of using trifluralin in vitro chromosome doubling in cabbage following microspore culture (Rudolf et al. 1999; Yuan et al. 2015) for double haploid induction. Autopolyploid induction in somatic tissues can raise new beneficial traits such as biotic or abiotic resistance, higher yield, higher glucosinolate content, and so on, due to either increased gene expression as a result of double sets of chromosomes or by gene redundancy promoting a buffering effect of deleterious recessive alleles and maintain the high level of heterozygosity (reviewed by Sattler et al. 2016). But lack of standardized precise methodology for in vitro polyploidization and clonal propagation limits the application of this technique in in vitro chromosome doubling in diploid tissues. The success of the technique relies on several variables, such as crop species, cultivar, explant, type of antimitotic agent and its concentration, treatment duration, application method, and so on. It is worth studying the influence of trifluralin, on the post-in vitro culture development of head cabbage and its effect on ploidy induction. As a starting step towards developing a successful in vitro protocol for polyploid induction using trifluralin as an antimitotic agent, in this study, we focused on examining the post effect of trifluralin on in vitro culture development of head cabbage starting from different explants following different trifluralin treatments. This study can provide a background for further studies on establishing reliable in vitro culture protocol and selecting appropriate explant for polyploid induction using trifluralin for head cabbage.

Nowadays, lack of proper knowledge of in vitro polyploidization and successful in vitro regeneration system in cabbage causes problems in the practical application of the technique in cabbage breeding. Mainly, in vitro chromosome doubling in crop plants is achieved using haploids (Dhooghe et al. 2011). As mitotic polyploidization has shown induction of important morphological and physiological changes in many crops including some Brassica species (Zhang et al. 2014, Marfil et al. 2018; Cheng et al. 2019), it is worth studying the effect and efficiency of trifluralin on in vitro cabbage culture morphogenesis and polyploidy induction. To our knowledge, this is the first report on the post effect of trifluralin on cabbage in vitro.

Among the three types of explants tested in this study, hypocotyl and calli showed a poor response for in vitro regeneration after being exposed to the trifluralin compared to the control proving the challenge of selecting appropriate explant for the successful in vitro organogenesis in conditions providing polyploid induction. In this experiment, we were not successful in shoot production from trifluralin-treated hypocotyl explant. After the trifluralin treatment, hypocotyls turned white-brown and died within one week on MS shoot induction medium. The mortality rate of the treated hypocotyls varied among cultivars as Z > S > M > F >A > DC6. Zeus showed zero survival rate of the hypocotyl (data is not presented). From surviving hypocotyls following the trifluralin treatment, some cultivars showed no further growth induction from hypocotyls but remained green and alive. Cultivars that showed some shoot induction either directly from the cut ends (Fig. 1), or from calli formed at the cut ends, did not grow into elongating shoots even after several transfers into fresh media either shoot induction or shoot development media.

Figure 1

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.

Figure 2

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 Hypericum and Rosa, respectively. Too low concentration and exposure conditions could be not effective but too strong conditions might not be lethal. Understanding the interaction between concentration and exposure time is important to achieve success. In this experiment, a significant interaction between treatment conditions, concentration, and genotype on seed germination and root induction was observed. This provides a basic platform for further studies using seeds as the starting material for ploidy induction in cabbage.

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 in vitro derived plantlets from treated seeds was determined by flow cytometric analysis using young leaf tissues and none of the plants were polypoid (data is not presented). All the regenerated plants were either 2× or 2× + 4×. There are reports on using trifluralin as a ploidy inducing agent from different explants of different species – in seedlings of Rhododendron (Eeckhaut et al. 2002), nodal segments in the family Rosaceae (Khosravi et al. 2008), nodes and shoots of Cucumis sativa (Ebrahimzadeh et al. 2018), embryos of Spathiphyllum wallsii (Eeckhaut et al. 2004), or in microspores of cabbage (Rudolf et al. 1999; Yuan et al. 2015; Li et al. 2020). There are no reports on using cabbage seeds as explants in vitro conditions. Kumar and Dwivedi (2014) used inbred seeds of cultivar B. campestris IC363713 to induce polyploids ex vitro employing colchicine, but they were unable to achieve any polyploids. Niimi et al. (2015) treated seeds and in vitro germinated seedlings of B. oleracea var. acephala (kale) with colchicine or amiprophos-methyl. They achieved the high frequency of tetraploids (24%) from APM (9 mg·L⁻¹ for 24 hours) treated seeds. In our study as well, we thought the seeds were a hopeful starting material for in vitro culture development in trifluralin mediated cabbage polyploid induction studies.

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 in vitro protocol usable for producing cabbage polyploids using trifluralin. To maximize the success of in vitro culture, namely organogenesis together with efficient ploidy induction, the careful optimization of treatment conditions and culture protocol for individual cabbage cultivar and a better understanding of the performance of each cultivar is required.