The effect of polyamines and silver thiosulphate on micropropagation of date palm followed by genetic stability assessment

There are some limitations in date palm micropropagation. These include low multiplication efficiency, low rooting rate, and high mortality experienced by in vitro raised plantlets during laboratory to soil transfer. The objective of the study was to determine the effect of the polyamines and Silver Thiosulphate (STS) on the enhancement of shoot multiplication and genetic stability of in vitro cultures of date palm cultivar Quntar. Media supplemented with 75 mg L−1 SPD in combination with 10 mg L−1 STS gave the highest percentage of callus producing buds (83.34%) and average bud formation (16.3) per jar. The addition of PUT and STS to the medium was most effective on root formation and the number of roots per shoot, where the best result, 91.67% and 6.37 roots per shoot, respectively, were obtained using 75 mg L−1 PUT and 10 mg L−1 STS, resulting in fast-growing plantlets during acclimatization phase, reaching 80% of plant survival. The genetic fidelity assessment of plants derived from micropropagation was confirmed by RAPD analysis. Four operon primers were used, and all of them showed amplified unambiguous (OPA02, OPC-04, OPD-07, and OPE-15). All generated bands were monomorphic and had no variation among the tissue culture-derived plants tested. Accordingly, these results indicate that adding polyamines and silver thiosulfate to the nutrient medium of date palm cv. Quntar was beneficial to improving shoot organogenesis, rooting, and production of genetically stable date palm plants.


Introduction
The date palm (Phoenix dactylifera L.), a tree of the palm family (Arecaceae), is cultivated for its sweet, edible fruits. The date palm has been prized from the ancient times and may have originated in ancient Mesopotamia, now known as Iraq (Wrigley 1995). The date palm can be propagated either from seeds or from offshoots. When plants are grown from seeds, about half of the palms turn to be males, which can be identified only upon flowering. Moreover, the plants obtained through seeds are genetically heterogeneous. Consequently, for uniformity of the orchards, date palms are propagated through offshoots only. Due to few offshoots that trees produce, the date palm is one case that requires serious attention for rapid vegetative propagation. Micropropagation technology has overcome these problems and provides uniform and good quality planting material to establish largescale plantations (Ibrahim et al. 2013;Al-Mayahi et al. 2018;Al-Mayahi and Ali 2021). The success of plant tissue culture largely depends on selecting the right culture medium. Nutritional requirements for optimal tissue growth in vitro may vary with the species. Even tissues from different parts of a plant may have different requirements for satisfactory growth; it is essential to work out a medium that will fulfill the specific requirements of that tissue (Al-Mayahi 2019, 2020. Organogenesis is one of the growth pathways through which somatic embryos can be stimulated to differentiation. Studies have shown that successful organogenesis can be achieved through the appropriate establishment of culture medium components, appropriate explant selection, and 1 3 124 Page 2 of 12 control of the physical environment (Thorpe 2007). It is clear from the literature that polyamines have an important role in plant tissue culture, as their use has expanded in recent years, especially in stimulating and multiplying shoots (Dey et al. 2019;Muhusen et al. 2020;Rakesh et al. 2021). Polyamines are low-molecular-weight organic cations essential for tissue growth and development due to their role in cell proliferation, signal transduction, and protein synthesis (Rakesh et al. 2021). Studies have shown that adding polyamines to culture media can enhance shoot growth, callus induction, and root regeneration (Tang and Newton 2005;Thiruvengadam et al. 2012). Chae (2016) reported enhanced plant regeneration of Echinacea angustifolia DC plants in vitro in the presence of polyamines. Kiełkowska and Adamus (2021) reported that putrescine "PUT" and spermidine"SPD" had a beneficial effect on the mitotic activity of cultured cells, which further affected the plant regeneration process. In vitro propagation has limitations, especially when the accumulation of ethylene in culture containers is severe and the genotypes exhibit sensibility to this phytohormone (Ievinsh et al. 2000). Studies have shown that externally applied polyamines can inhibit ethylene production in the culture medium (Panizzaaa et al. 1993;Laukkanen and Sarjala 1997). Thus it is likely to affect growth and development in such systems. The ethylene effects on in vitro morphogenesis are not fully understood, but the role of this hormone in senescence has been widely reported (Kumar et al. 1998;Wang et al. 2002). Senescence is a natural phenomenon related to ethylene and oxidative stress (Wang et al. 2002). The reactive oxygen species (ROS) are toxic molecules naturally produced as a result of aerobic metabolism, and therefore they should be rapidly and efficiently scavenged by antioxidant systems (Mittler 2002). Meanwhile, ethylene inhibitors can be used as a stimulator to promote callus induction and shoot regeneration (Al-Mayahi 2010). Roh et al. (2012) also reported that Silver Thiosulphate (STS) was more effective than AgNO 3 on shoot regeneration from cotyledon and hypocotyl explants of Brassic napus. The acclimatization process is crucial in the micropropagation process of date palm plantlets. One of the main obstacles to applying micropropagation technology is the high mortality rate during transfer to the soil. PAs in general and PUT in particular are nitrogen sources that have an anti-stress influence on stressed plants (Chen et al. 2019). Ethylene produced by plant tissues grown in vitro may accumulate in large quantities in culture vessels, thus potentially affecting growth and development. The beneficial effect of STS depends on the function of silver ion acting as an ethylene antagonist (playing as an inhibitor of ethylene biosynthesis) (Sharaf et al. 2012). Maintaining the true-totype nature of in vitro propagated plants in commercial and marketing processes is crucial for upholding certain agronomic and horticultural traits when using elite genotypes (Alizadeh et al. 2015). Nevertheless, plant micropropagation technology has a phenotypic and genetic variation of propagated plants known as somaclonal variation (Larkin and Scowcroft 1981). Out of various molecular markers used to evaluate in vitro regenerated plants' genetic fidelity, RAPD is one of the most simple, quick and cost-effective methods and require only small amounts of DNA (Chaudhary et al. 2015). Srivashtav et al. (2010) suggested that RAPD markers are more efficient than ISSR for assessing genetic variation in date palms. Several researchers used RAPD technique to examine genetic variability. It is an efficient and reliable technique for screening true to types of nature of tissue culture-derived plants (Moghaieb et al. 2011;Devi et al. 2013;Chaudhary et al. 2015). This study aimed to explore the effects of polyamines (putrescine "PUT" and spermidine "SPD") and Silver Thiosulphate (STS) supplementation on the performance of the growth date palm 'Quntar' and to determine the impact of these compounds on genetic stability for tissue cultured in vitro.

Materials and methods
The experiments of this study were carried out in the date palm micropropagation laboratory for the Date Palm Research Center at Basrah University, Basrah, Iraq. Young offshoots (2-3 years old) of date palm cv. Quntar were detached from the mother palm (Fig. 1a). Outer leaves and fibrous tissues at their bases were removed gradually until the shoot tip zone was exposed (Fig. 1b). Sheathing leaf base enclosing the very young leaves of the heart of the offshoot was left in place to protect it from disinfection solutions. The explants were taken and kept in antioxidant solution (100 mg L −1 ascorbic acid 150 mg L −1 citric acid). Sterilization of explants was performed using 70% ethanol for 1 min and 2.5% sodium hypochlorite for 20 min. Explants were then rinsed three times with sterile distilled water. The apical buds were sectioned longitudinally into four parts. In order to induce callus induction, explants were cultured on the MS basal medium (Murashige and Skoog 1962). Culture media was prepared from MS medium salts (4.43 g L −1 ) mixture containing the macronutrients and micronutrients. It was combined with Gamborg's vitamins and supplemented with 3 mg L −1 6-(dimethylallyl amino) purine (2iP), 30 mg L −1 , naphthalene acetic acid (NAA), 1.5 g L −1 activated charcoal, 30 g L −1 sucrose, 2.0 g L −1 activated charcoal, and solidified with agar-agar at 6.0 g L −1 were used. The pH of the medium was adjusted to 5.7 with 0.1N NaOH or HCl, before the addition of agar. Cultures were kept under complete darkness at 27 ± 2 °C. (Fig. 1c). The cultures were transferred to fresh media, with the same composition every 6 weeks until callus induction (Fig. 1d). For multiplication, the callus was transferred to MS media supplemented with 0.5 g L −1 activated charcoal, 6.0 mg L −1 NAA and 2.0 mg L −1 2iP. To study the effects of polyamine type on the multiplication of buds, the callus was divided and subcultured on shoot induction media supplemented as mentioned above, except for the growth regulators concentrations i.e., 1 mg L −1 (NAA) and 3.0 mg L −1 (2iP). It was also supplemented with Putrescine (PUT) and spermidine (SPD) in concentrations (0.0, 25, 75, and 150 mg L −1 ). Media were dispensed into culture jars and sterilized by autoclaving at 121 °C temperature and 1.04 kg cm −2 pressure for 20 min. All the cultures were incubated at 27 ± 2 °C and irradiated for 16 h with a diffuse light of daylight fluorescent lamps. Based on the result of our previous experiment in this study, the appropriate type of polyamine was selected in combination with silver thiosulphate (STS). To study the effects of these two compounds on enhancing organogenesis of shoots from callus, and shoot multiplication. MS medium was modified with different concentrations of SPD (0.0, 25, 75, and 150 mg L −1 ) in combination with STS (0, 5, 10, and 15 mg L −1 ) to study their effects on the percentage of shoot regeneration (%), and shoot number per jar.
The results of the experiments regarding the percentage of shoot regeneration and shoot number per jar were evaluated 12 week after the inoculation of callus on the media. Treatments were consisted of 12 types of media as shown in Table 1 Optimization of rooting and plant acclimatization Shoot clusters with no visible signs of root formation were collected in the elongation stage; the shoots were separated individually and cultured on rooting medium consisted of MS medium combined with Gamborg's vitamins and supplemented with 30 mg L −1 sucrose, 7.0 mg L −1 agar, 0.5 mg L −1 NAA and 0.5 g L −1 activated charcoal. The media were supplemented with two polyamines, PUT and SPD, in concentrations (0.0, 25, 75 and 150 mg L −1 ). The cultures were maintained at 16 h photoperiod, 25 ± 1 °C temperature and at16 h photoperiod at 25 ± 1 °C and irradiance of 13.5 µmol m −2 s −1 provided by cool white fluorescent tubes. Each treatment included 12 replicated jars. The percentage of root induction and root number per shoot were evaluated 6 weeks after the inoculation of shoots on the media. Based on the result of our previous experiment in this study, the appropriate type of polyamine was selected in combination with Silver Thiosulphate (STS) to study their effects on the rooting percentage (%) and root number. The rooting percentage was calculated as the number of shoots forming roots out of the total number of shoots cultured. Culture media were supplemented with different concentrations of PUT (0.0, 25, 75, and 150 mg L −1 ) in combination with Silver Thiosulphate (STS) at four concentrations (0, 5, 10, and 15 mg L −1 ). Each treatment included 12 replicated jars, incubated at room temperature 25 ± 2 °C, with a 16 h white florescent light photoperiod. The percentage of root induction and root number per shoot were evaluated six weeks after the inoculation of shoots on the media. There were ten replicates of each treatment, as shown in Table 2.

Acclimatization stage
For acclimatization, well-developed plantlets were gently washed with tap water to remove the remnants of agar. Then, the plantlets were washed with distilled water and treated with fungicide (Benlate 500 mg L −1 ) for 20 min and

Genetic stability of regenerated palnts
In order to study the genetic similarities, several regenerated plantlets were analyzed at the molecular levels using RAPD analysis.

RAPD analysis
Total genomic deoxyribonucleic acid (DNA) was isolated from regenerated date palm plantlets using the CTAB method described in Rogers and Bendich (1985). Polymerase chain reaction (PCR) reactions were conducted using a set of four arbitrary 4-mer primers (Operon Technology, Inc., Alameda, CA, USA). These primers and their sequences are presented in Table 3.

The PCR mixture
The reaction mixture (20 μl) contained 10 ng DNA, 200 μM deoxynucleotide riphosphates (dNTPs), 1 μM primer, 0.5 units of Red Hot Taq polymerase (AB-gene Housse, UK) and 10-X Taq polymerase buffer (AB-gene Housse, UK  (Adawy et al. 2004). The amplification products were separated in 1% (w/v) agarose gel in 1X Tris/Borate/Ethylenediaminetetraacetic_acid (TBE) buffer and visualized by staining with ethidium bromide. The reproducibility of DNA profiles was determined by replicating all RAPD reactions at least three times using DNA markers. The primers were evaluated from a wise pair comparison for the proportion of shared bands amplified (Nei 1978). The similarity coefficient was calculated by using the statistical software package STATISTICA-SPSS (Stat Soft Inc).

Experimental design and statistical analysis
The experiments were carried out using a completely randomized design(CRD). Data were analyzed using variance (ANOVA) analysis using Statistical Package for Social Sciences (SPSS) software version 20. Treatment means were compared using the least significant difference (LSD) at the P < 0.05 level.

Shoot induction and multiplication
Callus tissues showed significant variations in their response percentage, and Callus tissues in medium containing SPD at 75 mg L −1 showed a better response rate (73.34%) of callus producing shoots and average shoots number (9.18 shoots/ jar) (Table 4). Without polyamines, the control medium recorded the lowest response with the lowest number of buds (26.67%; 2.75), respectively. Results showed that when SPD was added 75 mg L −1 to the culture medium, the highest response percentage of callus producing buds with the highest number of shoots was recorded (73.34% and 9.18, respectively). From the results of our current study (Table 5), it was evidenced that the response percentage of callus tissue cultures producing buds with the number of buds increased with increasing concentration of polyamines used up to 75 mg L −1 and then decreased. The percentage of callus producing shoots and the number of shoots per jar also increased with the increase STS concentrations from 0 to 10 mg L −1 proportional to the concentration in the medium, but after that decreased with increasing STS concentrations. The combination between SPD and STS application had the highest response percentage and number of shoots, compared with treatments with no additives or one additive alone (Tables 4, 5). The highest response percentage and numbers of shoots (83.3% and 16.3) were obtained on the  media supplemented with 75 mg L −1 SPD and 10 mg L −1 STS, respectively (Fig. 2g).

Rooting and plantlets induction
From the data presented in Table (6), adding two types of polyamines separately at the studied concentrations to the nutrient medium improved the percentage of rooting and the number of roots cultured for date palm cv. Quntar. The highest significant value of percentage of rooting and the number of roots/shoot was obtained at (75 mg L −1 ) for each polyamine compound studied. The data showed that when PUT was added to the culture medium, the highest significant value was recorded, increasing the percentage of rooting and the number of roots/ shoot (80% and 5.75), respectively. A high concentration of 150 mg L −1 PUT positively affects the length of roots as compared with the other treatments. The combination between PUT and STS application had the highest percentage of rooting and the number of roots/ shoot, compared with treatments with no additives or a single additive alone (Tables 6, 7). The highest percentage of rooting and the number of roots/shoot (91,67 and 6.37) were obtained on the media supplemented with 75 mg L −1 PUT and 10 mg L −1 STS, respectively (Fig. 3). While the high concentration of 150 mg L −1 of PUT with all concentrations of STS positively affects root length compared to other treatments.

Acclimatization
The obtained data (Fig. 4) showed that adding polyamines PUT to silver thiosulfate (STS) improved the survival percentage. Maximum survivability was noticed for plants cultured in media containing both 75 PUT + 10 STS (Fig. 5), followed by plants cultured in media containing 75 PUT + 5 STS. The media did not contain STS, and the addition of PUT or STS at high concentrations was not suitable for date palm acclimation, where the survival percentage was low.

RAPD analysis
In this study, we regenerated plants from callus tissues with polyamine and silver thiosulfate STS; hence it becomes necessary to check the genetic stability of the regenerated plant. Random amplified polymorphic DNA markers (RAPD) were used in the present study under the influence of different treatments (Fig. 6). The results showed the genetic stability of in vitro propagated plants. The PCR amplification results showed a monomeric band in both the in vitro derived date palm plants and the mother plants of all primer pairs tested. RAPD analysis micropropagated plant (P. dactyliferaL cv. Quntar) indicated a profile similar to that of the control group that clearly showed the genetic stability of those plants (Fig. 6) and the fidelity of the in vitro propagation protocol to produce true-to-type date palm plants, indicating that the use of polyamine and STS during micropropagation phases caused no variation in the plants of this date palm cv. Quntar.

Discussion
Although shoot induction occurs, achieving shoot, elongation, and rooting are challenging in date palm (Al-Mayahi 2021a). Shoot organogenesis depends on many factors, such as culture medium composition and culture conditions (Al-Mayahi 2016, 2020. This study evaluated the effect of medium composition on shoot organogenesis. Our experiment indicates that using polyamines (PAs) with STS plays a synergistic role in promoting shoot formation from callus tissues of date palm in vitro. SPD with STS increased the regeneration frequency of date palm shoots in vitro. The addition of polyamines to the culture medium was influential in the regeneration of shoots. The best type and  concentration of polyamines used was SPD at 75 mg L −1 . PAs play a major role in cell division, plant growth, and development (Mattoo et al. 2010). It has been shown that PAs interact with plant hormones, act as PGR substances or secondary hormonal messengers, and as carbon and nitrogen storage in culture tissues (Couée et al. 2004). Furthermore,  PAs carry amino groups capable of interacting with macromolecules, such as nucleic acids, proteins, phospholipids, and cell wall components, and may have different effects on the culture medium (Takahashi and Kakei 2010;Tiburcio et al. 2014). Shoots multiplication helps increase the number of plantlets achieved through hormonal combination and PAs (Dey et al. 2019). The positive effect of polyamines on shoot regeneration can be attributed to their stimulatory effect on cell division (Bais and Ravishankar 2002). It has been suggested that regeneration and differentiation can be significantly improved by applying putrescine in date palm (Muhsen et al. 2020). It has been advocated that shoot regeneration and differentiation can be significantly improved by using PAs with ethylene inhibitors (Park et al. 2012). In the present study, the influence of ethylene inhibitor STS on in vitro culture of date palm was investigated. According to the results obtained, using STS in culture media can enhance the ability of date palm callus tissues to give the highest response percentage of shoots and shoot numbers. Silver thiosulphate is a suitable candidate for use in Quntar cultivar for the regeneration and multiplication of shoots and the rooting of plants. Although this response depends on the concentration of STS used. The highest percentage of callus producing buds and shoot number was achieved on media supplemented with 10 mg L −1 STS. High concentrations of STS do not have an important positive role in bud production. However, a medium without PAs and STS is the least effective for organogenesis (Table 1, 2). Ethylene produced by plant tissues grown in vitro may accumulate in large quantities in culture vessels, thus potentially affecting growth and development. This may be due to the role of silver ions in overcoming the action and metabolism of ethylene. Several studies support that ethylene affects callus growth and plant regeneration in vitro (Saiprasad and Raghuveer 2007;Sarropoulou et al. 2016). Sridhar et al. (2011) reported that STS significantly increased the shoot regeneration response and average buds in Solanum nigrum. Similar to our results, this finding is in a harmony with the results of Thiruvengadam and Chung (2015) who reported a positive correlation between SPD and shoot regeneration in gherkin(Cucumis anguria L.). However, the addition of SPD (75 mg L −1 ) in MS containing STS (10 mg L −1 ) produced a higher percentage of response as well as the number of shoots/jar when compared to STS alone (Table 5).
In Cucumis sativus (Vasudevan et al. 2008) and Withania somnifera (Sivanandhan and Salammal 2011), SPD supplementation of the culture medium improved shoot regeneration compared to putrescine as observed in the current study.
Our results are in agreement with previously reported results showing the stimulative role of PAs or STS in organogenesis in many plants (Bader and Khierallah 2009;Park et al. 2012;Arun et al. 2014;Roh et al. 2012;Muhusen et al. 2020). The number, length, and development of roots are essential in the in-vitro development with micropropagation. PAs play a vital role in rooting. The highest response percentage and numbers of roots were obtained on the media supplemented with 75 mg L −1 PUT and 10 mg L −1 STS. Although a high concentration of 150 mg L −1 PAs positively affects the length of roots compared to the other treatments, it has no significant effect on rooting percentage and the number of the roots. PAs are involved in various cellular and physiological pathways and cycles that promote root growth, proving essential in differentiation (Tiburcio et al. 2014). PAs play a major role in cell division and different morphogenetic processes, including rooting (Kiełkowska and Adamus 2021).
Our results indicate that PUT improved rooting efficiency, whereas spermidine showed less response to root induction. Denaxa et al. (2014) reported that PUT improved the rooting response of difficult-to-root 'Kalamata' olive cultivar, compared with SPD, which failed to promote rooting. Endogenous PUT is considered a marker of root induction in vitro. Its catabolism could be the basis for root growth by providing H 2 O 2 (Neves et al. 2002). It has previously been shown that PUT to MS media increases endogenous PUT accumulation to promote root induction and growth (Hausman et al. 1995). PUT also acts as a second messenger, correlating with the peak of root mitotic activity (Tiburcio et al. 1989). Similarly, PUT induced root induction in Pinus virginiana (Tang and Newton 2005). The encouragement of palm plants during the rooting stage by STS may be due to the unique function of silver, which appears to be unique among the heavy metals that play an inhibitory role in ethylene biosynthesis. This result is in harmony with Sharaf et al. (2012), who reported a positive correlation between response to rooting and STS. Roh et al. (2012) reported that the medium supplemented with STS compound encouraged roots cultures to elongate and proliferate. Similar results were obtained by Harathi and Naidu (2016), who suggested that adding ethylene inhibitor to the culture medium along with an auxin significantly augmented the induction of roots.
Micropropagation cannot be considered completely successful unless complete genetic fidelity is maintained. The regenerated plants from tissue culture were checked for their genetic stability using RAPD primers. RAPD has been extensively used in genetic variation experiments in date palm plants derived from tissue culture (Saker et al. 2000;Moghaieb et al. 2011). Four primers were selected based on the amplified bands' quantity, quality, and reproducibility. All bands matched perfectly with the DNA of the field donor plant. The detected bands were 100% monomorphic, indicating that the use of PAs and the STS during micropropagation phases caused no variation in the tissue culture-derived plants of this date palm genotype. (The resultant clones are true-to type of the selected genotype). PAs carry positive charges on nitrogen atoms; this helps electrostatic attraction between DNA, RNA, proteins, and phospholipids. Hence, PAs play a role in membrane fluidity, signal transduction, elicitation, RNA processing, chromatin remodelling, etc. (Baron and Stasolla 2008). Shenoy and Vasil (1992) reported that micropropagation through explants containing organized meristem is generally associated with a low risk of genetic instability. The culture conditions used to achieve plant regeneration from tissue where meristems are already present are less aggressive than those usually needed to induce shoots from differentiation. This result is in agreement with that previously reported by El-Bahr et al. (2019). Similar results were obtained by some other authors (Abdol vand et al. 2018;Al-Mayahi 2021c), who used molecular markers to confirm the genetic stability of micropropagated date palm plantlets.
The banding pattern analysis confirmed no somaclonal variation and, therefore, the reliability of the micropropagation protocol for producing true-to-type plantlets of date palm cv. Quntar on a mass scale.

Conclusion
This study provides an efficient in vitro propagation protocol for producing genetically uniform date palm plants.
Our study indicated that SPD alone or in combination with STS plays a synergistic role in improving shoot regeneration from callus tissues of date palm cv. Quntar in vitro. Also, particular emphasis should be given on PUT, which in combination with STS, in the rooting medium, was essential in stimulating a high rooting percentage with high quality of roots; thereby, resulting in fast-growing plantlets during acclimatization phase, reaching 90% of plant survival. On the other hand, no genetic variation was observed by the four RAPD primers tested. The in vitro micropropagation protocol developed in this research could be used for the largescale production of genetically stable date palm.cv. Quntar.