Influences of Thidiazuron Concentrations and Exposure Duration on Axillary Shoot Proliferation of Faba Bean [Vicia faba (L.)] Genotypes

Faba bean (Vicia faba L.; Fabaceae) is an excellent source of protein and is an important legume crop for human food and animal feed (Paul and Gupta, 2021). As a leguminous species, faba bean also plays a significant role in the organic fixation of nitrogen from the atmosphere (Adak and Kibritci, 2016; Jelenić et al., 2000). Despite the significance of this crop, there has been little progress in its improvement because of its high out-crossing rate (as high as 50%). In addition, this crop shows yield instability and, in general, a low level of genetic diversity which restricted its genetic improvement. Faba bean is a recalcitrant species for in vitro regeneration (Khalafalla and Hattori, 2000). It exhibits poor regeneration from callus and tissue cultures are often inhibited by the release of phenolic compounds from the tissues. For these reasons, it is difficult to conduct in vitro studies on this crop (Böttinger et al., 2001). Faba bean is one of the least-studied legumes and only a few reports describe in vitro culture. Previous tissue culture studies used individual seedlings (Busse, 1986), micro-cuttings and auxiliary buds (Selva et al., 1989), cotyledonary nodes (Khalafalla and Hattori, 1999) and protoplasts (Tegeder et al., 1995) as explants. Anwar et al., (2011) used cotyledon explants with half embryonic axis in faba bean cultivars for indirect regeneration system (Adventitious shoot proliferation). These studies correlated regeneration efficiency to the culture conditions, such as the effect of hormones, the composition of the medium, or other physical conditions. Moreover, genotypes have shown to impact the faba bean regeneration. Ahmed et al., (2020) evaluated the growth, morphological changes and production of phenolics in the in vitro plantlets of five Egyptian faba bean cultivars. The authors revealed that cultivars ‘Nubaria 2’ and ‘Skha 3’ had the highest growth parameters and regeneration frequency (85.3% and 78.6%), respectively.
       
Thidiazuron (TDZ) is a powerful and potent synthetic growth regulator that has a wide array of effects on plant growth and development. However, prolonged exposure to TDZ can have negative effects on growing cultures and transferring the cultures to a medium without TDZ can alleviate these effects and promote further shoot proliferation and multiplication (Dewir et al., 2018). TDZ has been found to have both positive and negative effects on the growth and development of faba bean in vitro. TDZ has been shown to be effective in inducing adventitious bud formation and promoting shoot proliferation in faba bean explants (Anwar et al., 2011). However, high concentrations of TDZ can be toxic to plantlets (Khalafalla and Hattori, 1999). Additionally, the use of TDZ in faba bean tissue culture has been associated with morphological, physiological and cytogenetic abnormalities (Tegeder et al., 1995). Despite these drawbacks, TDZ has been successfully used in the regeneration and genetic improvement of faba bean through micropropagation and transformation protocols.
       
Overall, TDZ can be a valuable tool for the in vitro manipulation of faba bean, but its application should be carefully optimized to avoid negative effects (Dewir et al., 2018). An indirect somatic embryogenesis regeneration system of faba bean was reported by Griga et al., (1987). Reproducible regeneration was reported for faba bean using single cotyledon explants with half embryonal axis and cotyledonary node (Khalafalla and Hattori, 2000; Anwar et al., 2011) indicating that TDZ allows for successful micropropagation of faba bean, which is essential for future genetic improvement of faba bean. However, these studies also reported that the response of faba bean genotype differ among faba bean genotypes. Therefore, the objectives of this study were to screen the responsive faba bean genotypes of different sizes to different TDZ concentrations and exposure durations for their axillary shoot proliferation in vitro.

Plant materials, surface sterilization and culture establishment
 
This study was conducted during the year 2020 at the plant tissue culture laboratory, College of Food and Agricultural Sciences, King Saud University. Ten genotypes of faba bean (Vicia faba L.) were obtained from ICARDA, Sudan, Spain, Egypt and Saudi Arabia. The genotypes name, pedigree, origin, seed type and agronomic features are listed in Fig 1A and Table 1. The selection criteria for those genotypes are based on origin and seed type (large, medium and small), to represent the Saudi landraces, Middle East, North Africa and Europe. Seeds were washed with tab water, then surface sterilized with 70% (v/v) ethanol for 30 seconds, followed by soaking in 20% (v/v) commercial CloroxTM (5.2% sodium hypochlorite) solution for 15 min. The seeds were washed five times with sterile distilled water under aseptic conditions prior to soaking overnight. The imbibed seeds were de-coated and cultured for 6 days into Petri dishes that contained 25 mL MS (Murashige and Skoog, 1962) medium without PGRs for their germination under dark conditions (Fig 1B).  Prior to autoclaving at 121°C for 20 minutes, the pH of all the medium variants was adjusted to 5.7. Additionally, the medium was gelled with 0.7% (w/v) Sigma agar-agar. All the cultures were incubated at 25±2°C under a 16 h photoperiod provided by cool-white fluorescent tubes. The intensity of the lights was set at 35 µmol m^”2 s^”1 photosynthetic photon flux (PPF). The length of shoots and roots was recorded for Luz (large seeded); ILB4347 (medium seeded) and Triple white (small seeded). All measurements were obtained from 10 randomly selected explants.
 

 

 
Effect of explant type (cotyledonary node with and without single cotyledon) and light regime  on shoot multiplication and growth of faba bean
 
De-coated seeds (Fig 2A,B) of three genotypes (Luz, ILB 4347 and Triple White) were cultured on basal MS medium supplemented with 30 g/L sucrose and incubated under three different light regimes (T1: 6 days in dark, T2: 3 days in dark followed by 3 days in light and T3: 6 days in light) and two different explants (cotyledonary nodes explant consisting of 5 mm hypocotyl and epicotyl tissues with or without single cotyledon) as described in Fig 2 C,D. The explants were then cultured onto MS medium without PGRs for two weeks. The illumination was set at 16:8 photoperiod provided by cool white fluorescent tubes and light intensity of 100-150 μmol m^-2 s^-1 PPFD. The shoot growth and shoot multiplication responses were assessed following 2 weeks culture period whereby, number of axillary shoots developed per explant and shoot length were recorded. All measurements were obtained from 10 randomly selected explants.
 

 
Effect of thidiazuron concentration and exposure duration on axillary shoot multiplication of faba bean genotypes
 
For axillary shoot multiplication, cotyledonary node with single cotyledon explants of the ten genotypes (Table 1) were cultured in Magenta GA-7 culture vessels (77×77×97 mm; Sigma Chemical Co., St. Louis, Missouri, USA) that contained MS medium supplemented with 30 g L^”1 of sucrose and five different concertation of thidiazuron (TDZ; 2, 4, 6, 8 and 10 mM) and two different duration of exposure to TDZ at 10 or 20 days. The regenerated axillary shoots from explants were sub-cultured onto MS medium for 2 weeks without PGRs for their elongation. All cultures were incubated at 25±2°C under cool-white fluorescent tubes that provided a 16:8 h photoperiod and light intensity of 100-150 µmol m^-2 s^-1 PPFD. All measurements were obtained from 10 randomly selected explants.
 
Experimental design and data analysis
 
All experiments were conducted in a completely randomized design. The impact of the treatments was evaluated using Tukey’s multiple range test in SAS (version 9.4; SAS Institute, Inc., Cary, North Carolina, USA).

Effect of light regime and explant type on axillary shoot multiplication and growth of faba bean seedling
 
The seedling growth (shoot and root length) of three faba bean genotypes under three different light regimes are presented in Table 2 and Fig 3. The overall mean of shoot and root length showed that T1 treatment gave higher shoot and root length followed by T2 and T3 . It is noted that medium size seeded genotype ILB4347 showed the highest shoot and root length mean with 3.57 cm and 5.11 cm, respectively, as compared with large and small seeded genotypes. After that, the seedlings were used as source of explants by excising seedlings into half containing 5 mm hypocotyl and epicotyl tissues with or without single cotyledon. The two different explants were culture on MS basal media for 2 weeks. The average of number of shoot generated by each treatment is presented in Table 3. The explants with single cotyledon gave higher number of shoot than explant without single cotyledon with an average of 1.13 shoot (Fig 3B). On the basis of genotype used, ILB4347 on explant with single cotyledon generated the highest number of axillary shoot followed by Luz and Triple White. This result was positively correlated (r=0.657) with seed size (100-seed weight). The overall results showed that explant with single cotyledon treated by T1 treatment gave the highest axillary shoot with 1.22 shoot. Although, the value is not significantly different from the other light incubation treatments, but T1 treatment produced more axillary shoot regardless of explant source. Therefore, explant with single cotyledon and T1 treatment were used in the further experiment. Shoot length of each treatment was also measured and the results were presented in Table 4. The explants containing single cotyledon had longer shoot compared explants without cotyledon with 11.05 cm compared to 5.89.
 

 

 

 

 
Effect of TDZ concentrations and time of exposure (TE) on axillary shoot multiplication of faba bean genotypes
 
Effect of TDZ concentrations and time of exposure on shoot multiplication of ten faba bean genotypes is presented in Table 5. The overall mean of time exposure of TDZ showed that treatment of 20 days exposure had higher number of shoot compare to treatment 10 days exposure with 3.77 shoot/explant. Treatment of 10 mM TDZ after 20 days in culture generated the highest number of shoot/explant with 4.81 shoot/explant. On the basis of genotype used, the three most responsive genotype in tissue culture were ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot/explant, respectively. The clumps of multiple shoots were then cultured on MS without PGRs for shoot elongation (Fig 4A, 4B). Interestingly, the number of shoot/explant increased after 2 weeks in culture (Fig 4C) indicating remaining effect of TDZ in the explant. The shoot multiplication of ten faba bean genotypes after 2 weeks culture on MS free PGRs is presented in Table 5. The number of shoot/explant of genotype ILB4347 and Hassawi 2 increased to 6.26 and 6.10, respectively. Treatment of 8 mM TDZ after 20 days followed by 2 weeks on MS free PGRs generated the highest number of shoot/explant with 6.63 shoot/explant. Based on this screening, it was concluded that ILB4347 were the most responsive genotype in tissue culture and treatment 8 µM TDZ after 20 days followed by 2 weeks on MS free PGRs was the best treatment for shoot induction media.
 

 

       
Unlike other cytokinins, TDZ demonstrates resistance to endogenous cytokinin oxidase, resulting in its considerable stability in tissue culture (Mok et al., 1982). The metabolic rate of TDZ is notably sluggish, whereas zeatin is fully metabolized by plant tissues mere hours after its application (Mok and Mok, 1985). TDZ effectively suppresses the activity of cytokinin oxidase (Horgan et al., 1988; Hare et al., 1994), thereby leading to the accumulation of purine cytokinins in plant tissues. TDZ can therefore be utilized exclusively to facilitate distinct morphogenic and regeneration pathways. The appropriate and optimal concentration of TDZ is species-specific. TDZ enhances the synthesis of adenine-type cytokinins, modulates endogenous hormones and exhibits both auxin and cytokinin-like activities. Moreover, it promotes stress genes and leads to the production of ethylene and stress signaling molecules. TDZ can thus be used solely to achieve different morphogenic and regeneration pathways (Dewir et al., 2018).
       
The present study screened the most responsive faba bean genotypes in order to develop an efficient and effective protocol for shoot multiplication of faba bean in vitro. The current study tested two different explants which were cotyledonary node containing 5 mm hypocotyl and epicotyl tissues with or without single cotyledon. These explants were originated from seed which were germinated on three different light treatment. The investigations found that cotyledonary node with single cotyledon that incubated on 6 days in dark had the highest number of shoot. Therefore, this explant was used for screening the most responsive faba bean genotypes. The ten faba bean genotypes were screened under different TDZ concentrations and time of exposure to TDZ. The overall mean showed that genotypes of ILB4347 and Hassawi 2 were the most responsive genotypes in tissue culture. Based on this experiment, treatment of 8 mM TDZ for 20 days in culture obtained the highest number of shoot after subsequent culture in MS free PGRs for 2 weeks. This treatment also resulted the highest percentage of callus regeneration. Our results obtained 11 shoot per explant after 34 days in culture as compared with 5.9 shoot per explant after 45 days observed by Abdelwahd et al., (2008). Previous study by Anwar et al., (2011) demonstrated that TDZ (6 mM) in combination with 2-iP (10 mM) and kinetin (4 mM) was optimal for expansion of the meristematic zone followed by adventitious bud/shoot induction.
       
The effectiveness of TDZ in the in vitro propagation of various plant species within the Fabaceae family has been well established. Parveen and Shahzad (2010) detailed the in vitro propagation of Cassia sophera using cotyledonary node explants excised from axenic seedlings aged 21 days. All explants cultured on MS medium supplemented with different TDZ concentrations (0.1-10 mM) exhibited multiple shoot formation, with 2.5 mM concentration proving optimal for producing a maximum of 6.7 shoots per explant. Similarly, multiple shoots of Pterocarpus marsupium were induced from cotyledonary nodes of 18-day-old axenic seedlings on MS medium supplemented with 0.1-10 mM TDZ. Notably, a concentration of 0.4 mM TDZ resulted in the highest shoot regeneration frequency (90%) and number of shoots per explant (15.2) (Husain et al., 2007). In the case of Senna alata, a high number of nodes and shoots (8.3 and 12.6, respectively) were obtained by utilizing MS medium containing 1 mg/L TDZ and 0.1 mg/L NAA (Lara et al., 2022).
       
Significant advancements have been made in the field of tissue culture for recalcitrant plant species through the application of TDZ. The utilization of TDZ at lower concentrations within the range of nanomolar to a few micromolar has demonstrated the capacity to stimulate axillary shoot production, whereas elevated concentrations promote the development of callus and the propagation of adventitious shoots as noted by Huetteman and Preece (1993), Debnath (2018) and Vinoth and Ravindhran (2018). Various plant species exhibit a greater receptiveness to a wider range of TDZ concentrations and subculture cycles, as evidenced by the abundance of successful tissue culture protocols employing TDZ, wherein these plant species display normal growth. Nevertheless, the implementation of TDZ at low concentrations, pulse treatment and short period exposure durations are effective strategies to circumvent TDZ-induced abnormalities. In the case of Bactris gasipaes, a pulse treatment with a concentration of 0.36 µM TDZ for a duration of 14 days effectively mitigated the adverse effects brought about by prolonged exposure to TDZ (Graner et al., 2013).

The present study investigated the optimal formulation for axillary shoot multiplication in different faba bean genotypes. Light regime, genotype and TDZ concentration and duration of exposure significantly influenced axillary shoot multiplication of faba bean. TDZ at 8 µM concentration and exposure duration of 20 days resulted the highest the highest proliferation with 6.63 shoot per explant. Moreover, different faba bean genotypes displayed varied responses. The three most responsive genotype in tissue culture were medium sized seeds genotypes (ILB4347, Hassawi 2 and Misr 3 with 4.76, 4.40 and 4.15 shoot per explant, respectively.

The authors acknowledge Researchers Supporting Project number (RSP2024R375), King Saud University, Riyadh, Saudi Arabia.

All authors declared that there is no conflict of interest.