Impaginato 433 Adv. Hort. Sci., 2019 33(3): 433-439 DOI: 10.13128/ahs-23476 Efficient and easy micropropagation of Morus nigra and the influence of natural light on its acclimatization W.N. Duarte ¹ (*), C.A. Zanello ¹, J.C. Cardoso ² ¹ Master’s Graduate Program in Plant Production and Associated Bioprocesses, Centro de Ciências Agrárias, UFSCar, Araras, Brazil. ² Department of Biotechnology, Plant and Animal Production, Centro de Ciências Agrárias, UFSCar, Araras, Brazil. Key words: benzyladenine, black mulberry, greenhouse, growth room, plant growth regulators. Abstract: Micropropagation, which employs various plant growth regulators (PGRs) in the culture medium for the induction of multiple shoots as well as adventitious roots, is a widely-used technique for the propagation of the genus Morus. The main objective of the present study was to evaluate the effect of PGR-free culture medium on the quantitative and qualitative characteristics of the micropropagation of Morus nigra under the growth room and greenhouse environmental conditions. Although a higher rate of multiplication (4.7–5.2 shoots/explant) was obtained from the treatments using Benzyladenine (BA) as the PGR, the PGR-free culture medium also exhibited comparable multiplica- tion rate (4.1 shoots/explant) with a higher quality of shoots and without any symptoms of hyperhydricity. Furthermore, the use of PGR-free culture of M. nigra for in vitro propagation combined the steps of shoot multiplication and rooting phase using the same culture medium, further simplifying the process. In this study, the micro-shoots were also assessed for their in‐vitro rooting and acclimatization potential. The incubation of an in‐vitro culture of nodal explants in a controlled growth room for 28 days exhibited optimum response with about 90% rooting and 100% plantlet survival during the acclimatization phase. These results were better than those incubated under greenhouse con- ditions. 1. Introduction Morus nigra is an exotic plant of unknown origin, with wild popula- tions occurring in the Aegean Region of Turkey (Browicz, 2000). Its fruits are a rich source of phenolic compounds such as flavonoids (Arfan et al., 2012). Its propagation via seeds is limited due to a high degree of heterozy- gosity and a long period of sexual immaturity to reach fruit production (Vijayan, 2014). Mulberry (Morus sp.) is commercially propagated via the rooting of stem cuttings. However, in vitro clonal production of plantlets via micropropagation could also be achieved using MS medium supple- (*) Corresponding author: willnduarte@gmail.com Citation: DUARTE W.N., ZANELLO C.A., CARDOSO J.C., 2019 - Efficient and easy micropropagation of Morus nigra and the influence of natural light on its acclimatization. - Adv. Hort. Sci., 33(3): 433-439 Copyright: © 2019 Duarte W.N., Zanello C.A., Cardoso J.C. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 26 September 2018 Accepted for publication 24 April 2019 AHS Advances in Horticultural Science Short note http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2019 33(3): 433-439 434 mented with benzyladenine (BA) at concentrations of 0.5 to 2.5 mg L-1 for multiple shoot induction and indole butyric acid (IBA) or naphthalene acetic acid (NAA) at concentrations of 0.25 to 2.0 mg L-1 for root induction, in different species of this genus (Chitra and Padmaja, 2005; Ahmad et al., 2007; Ji et al., 2008; Chattopadhyay et al., 2011). Some protocols have also used gibberellic acid (GA3) for the elonga- tion of shoots (Gogoi et al., 2017). Although plant growth regulators (PGRs) such as BA, IBA, and NAA promote and regulate the in vitro development and improve the efficiency of micro- propagation, the addition of PGRs to the culture medium may also result in undesirable effects, vary- ing from the loss of quality of generated plantlets and phenotypic variations, to certain undesirable genetic mutations called somaclonal variations (Bairu et al., 2011). The control of environmental conditions in a growth chamber or culture room for in vitro propaga- tion is another requirement of commercial micro- propagation, which increases the costs of microprop- agated plantlets (Chen, 2016). Cardoso et al. (2013) suggested that the use of photoautotrophic micro- propagation following pre-acclimatization in a green- house using natural light could reduce the costs of this technique, along with enhanced in vitro plantlet quality. This study aimed to develop a simple protocol for the micropropagation of black mulberry (M. nigra) without the use of plant growth regulators (PGR-free micropropagation). The study also evaluated the use of in vitro pre-acclimatization under greenhouse con- ditions for the acclimatization of generated M. nigra explants. 2. Materials and Methods Establishment of in vitro cultures of Morus nigra Apical and axillary buds procured from mature (4- year-old) field-grown plants of M. nigra cv. ‘Portuguesa’ were used for the establishment of in vitro cultures. Young shoots of 1 to 2 cm length were immersed in 70% (v/v) ethyl alcohol for 30 sec, fol- lowed by surface steril ization in 40% sodium hypochlorite solution (containing 2.0-2.5% of active chlorine) for 15 min. After asepsis, the explants were thoroughly washed with sterile distilled water and trimmed further to a length of 3-5 mm using a scalpel blade, under a laminar airflow hood. The explants were then inoculated in flasks containing 30 mL MS medium (Murashige and Skoog, 1962) with half strength of macronutrients (½ MS) and supplement- ed with 30 g L-1 sucrose, 0.1 mg L-1 myo-inositol, 1.0 mg L-1 BA, and 0.1 mg L-1 NAA. The pH of the culture medium was adjusted to 5.8±0.05, after which 6.4 g L-1 agar-agar was added and the medium was auto- claved at 121°C and pressure of 1 kgf/cm² for 20 min. The culture flasks were incubated in a growth room with a light intensity of 2,500 lux (cold fluorescent lamps of 20 watts), 16-h photoperiod, and tempera- ture of 25±2°C. After in vitro establishment, the explants were maintained every 30 days by sub-culturing the nodal segments in ½MS culture medium without the addi- tion of plant growth regulators, until the numbers of segments obtained were sufficient to execute the experiments. Effect of different PGRs on in vitro shoot multiplica‐ tion The basal culture medium described for the in vitro establishment (½MS medium supplemented with 30 g L-1 sucrose, 0.1 mg L-1 myo-inositol) was used throughout the study, with variations in the types and concentrations of the most commonly used PGRs for the genus, viz., PGR-free; BA at 0.25, 0.50, and 1.0 mg L-1; Thidiazuron (TDZ) at 0.10 mg L-1; and GA3 at 3.0 mg L-1 (Vijayan, 2014). For the experiment, the nodal explants (1.0±0.2 cm) with one axillary bud were obtained from in vitro cultures previously grown on PGR-free ½MS medium for 30 days. The growth conditions were identical to those described previously. The experiments were conducted using a com- pletely randomized design (CRD), with five replicates (flasks) containing four nodal explants in each. After 30 days of incubation of the culture, growth parame- ters such as shoot length, number, and width of leaves, and multiplication rate (number of new nodal segments obtained from each inoculated explant) were evaluated for each treatment. All the measure- ments were carried out using a digital caliper. In vitro rooting and acclimatization The nodal explants (1.0±0.2 cm) containing one axillary bud were inoculated in flasks containing ½MS basal medium without PGRs. Eight treatments in a 2×4 factorial design, with two environmental condi- tions, i.e., culture room and greenhouse, and four different durations of in vitro culture (7, 14, 21, and 28 days) were used. The culture room conditions were identical to those described in the establish- ment phase, while the greenhouse was covered with Duarte et al. ‐ Efficient and easy micropropagation of Morus nigra 435 diffusion agricultural plastic sheets of 150 µm thick- ness, with a light intensity of 65000±3000 lux, and average temperature ranging from 28.1 to 29.5°C during the period of in vitro rooting and acclimatiza- tion phase. Each treatment comprised of four flasks containing five nodal segments in each. The in vitro assessments were conducted after each period of growth (7, 14, 21, or 28 days). The number of nodal segments with new sprouts and roots was the para- meter used for the evaluation. The field-based assessments were also conducted after 30 days of plant growth under greenhouse conditions on a com- mercial substrate consisting of peat (Pindstrup®, Denmark). The evaluation parameters included the height of plantlets, and the number and width of leaves. All the measurements were performed using a digital caliper. The obtained data were analyzed using analysis of variance (ANOVA) and the means were subjected to Tukey’s comparison post-test at 5% probability. The software ASSISTAT version 7.7 beta was used for the statistical analysis (Silva and Azevedo, 2006). 3. Results and Discussion Successful establishment of Morus nigra The development was initiated through the growth of shoots with fresh leaves after 14 days of inoculation, without any contamination or oxidation of explants. The first subculture was carried out after 30 days of inoculation and the shoots were multiplied until sufficient quantity of the explants was obtained for the ensuing micropropagation experiments. PGR‐free culture medium resulted in single‐step micropropagation and higher quality of plantlets The nodal segments cultured on PGR-free ½MS medium showed the best average height of plants (6.4 cm), with a good number (4.4) and width of leaves (1.8 cm). The addition of 0.25 mg L-1 and 1.0 mg L-1 BA increased the multiplication rate from 4.1 (PGR-free) to 4.7 and 5.24, respectively (Table 1). However, the addition of BA, even at the lowest concentration used in culture media (0.25 mg L-1), negatively affected the quantitative (height of plants and number and width of leaves) (Table 1) as well as qualitative parameters of regenerated shoots, show- ing callus formation at the basal cut ends and yellow- ish leaves (Fig. 1 A-C), compared with PGR-free medi- um (Fig. 1 F). These symptoms were more intense (yellowish-brown leaves) in case of TDZ, even at a low concentration of 0.1 mg L-1 (Fig. 1 D). The addition of GA3 resulted in a reduction in the number of leaves and axillary buds, leading to a lower multiplication rate (Table 1; Fig. 1 E) than the PGR-free medium. In most of the protocols related to in vitro micro- propagation of Morus sp., the addition of cytokinins Table 1 - Effects of culture medium with different PGRs on the in vitro development and multiplication of nodal segments of Morus nigra * Multiplication rate referring to the mean number of nodal segments obtained by each segment inoculated in vitro. Average values followed by the same letter in the column do not differ from each other by Tukey's test at 5% probability. Treatment Plantlet Leaves Height (cm) Multiplication rate* Number Width (cm) PGR-free 6.4 A 4.1 BC 4.5 A 1.8 A BA 0.25 3.5 B 4.7 AB 3.7 B 1.2 B BA 0.50 3.5 B 5.0 A 3.7 B 1.1 B BA 1.00 3.3 B 5.2 A 3.6 B 1.1 B GA3 3.6 B 2.2 D 2.4 C 1.2 B TDZ 2.3 C 3.6 C 3.4 B 1.1 B F value (PGRs) 18.24 22.66 7.86 16.59 Coefficient of variation (%) 38.3 26.4 30.8 26.6 Fig. 1 - Shoots of Morus nigra in different types and concentra- tions of plant growth regulators (PGRs): (A) 0.25 mg L-1 BA; (B) 0.50 mg L-1 BA; (C) 1.0 mg L-1 BA; (D) 0.10 mg L-1 TDZ; (E) 3.0 mg L-1 GA3. (F) PGRs-free MS½ medium. Bar = 1 cm. Adv. Hort. Sci., 2019 33(3): 433-439 436 to the culture medium had shown positive effects on the multiple shoot induction (Chattopadhyay et al., 2011; Lalitha Natarajan et al., 2013). Until now, no report was available on the use of PGR-free culture medium for the in vitro multiplication of Morus sp. Yadav et al. (1990) observed a high shoot multiplica- tion rate from shoot apex (4.2) and nodal explants (11.3) in M. nigra using MS medium supplemented with 1.0 mg L-1 BA. They also recorded a high shoot multiplication rate (6.3) with nodal explants using kinetin (KIN). Anis et al. (2003) recorded in vitro mul- tiplication rates ranging from 2.4 to 5.2 using 2.0 mg L-1 BA and 0.2 mg L-1 NAA in M. alba. On the other hand, although Akram and Aftab (2012) did not observe multiple shoot induction in M. macroura using either BA or KIN singly in MS medium, the sup- plementation of BA with IBA in the culture medium resulted in a high multiplication rate of 4.7 shoots/ explant. In the current study, the excision of shoot apical meristem (SAM) of M. nigra helped in breaking the dormancy of axillary buds in nodal explants cultured in vitro and increased the level of cytokinin. Excision of the SAM also caused a reduction in the endoge- nous levels of auxins and enhanced the expression of genes encoding Isopentenyl transferase (IPT), a key precursor enzyme for the biosynthesis of endoge- nous cytokinin. This increased the level of endoge- nous cytokinin in the axillary buds, resulting in stimu- lation of cell division and shoot development (Tanaka et al., 2006). The highly resistant nature of BA to the degradation activity of cytokinin oxidases explained the largely improved in vitro shoot productivity when this PGR was added to the culture medium (Ashikari et al., 2005). The use of PGR-free medium resulted in the pro- duction of shoots and roots without any supplemen- tation. However, Yadav et al. (1990) observed that IBA was more effective in the promotion of rooting in M. nigra as compared to IAA and NAA, and increased the number of roots from 1.2 (auxin-free) to 6.1 (0.25 mg L-1). These results are strikingly similar to our find- ings, as we also obtained only 1-2 roots per stem nodal segment without using auxins (data not shown). Nevertheless, although a lower number of roots were produced, even a single root was enough for the acclimatization of Morus nigra plantlets. The plants obtained from the culture medium containing BA exhibited some morphological alter- ations compared with those developed in the PGR- free medium. The abnormalities included difficulty in in vitro manipulation during subculture due to the breakage of brittle tissues, reduced internodal length, and yellowish leaf appearance. These symp- toms of hyperhydricity were observed irrespective of the BA concentration (Fig. 1A-C). The repeated sub- cultures on BA containing medium led to somaclonal variations in the micropropagated plants (Nasser and Mahmood, 2014). Nanism, hyperhydricity, and chlorosis were some of the abnormalities reported in the in vitro‐raised plants with continuous use of high concentrations of BA (Israeli et al., 1991; Kumar et al., 1999). In the current study, no morphological abnormality was observed in the obtained plantlets, even after 24 months with mensal subcultures, when PGR-free culture medium and nodal explants of 1.0±0.2 cm were used. Greenhouse pre‐acclimatization did not show optimal development of plantlets A notable difference was observed in the shoot development from in vitro nodal segments of mul- berry after seven days of inoculation and 21-28 days of growth in growth room and under greenhouse conditions, with about 100% of nodal explants pro- ducing new shoots in the growth room compared to only 80% under greenhouse conditions (Fig. 2 A). However, the highest impact of environmental condi- tions was observed on the rooting, where only 40% of the nodal segments produced roots in the green- house compared to 90% in growth room conditions (Fig. 2 B). Although up to 80% shoot regeneration from the nodal explants was recorded after 14 days of culture in greenhouse conditions, the rate later reduced to 70% and 40% after 21 and 28 days, respectively. This could be attributed to the death of plantlets after cultivation for 14 days in greenhouse conditions, possibly due to excessive ambient light intensity (above 68,000 lux), which caused maximum daytime temperatures reaching between 35.1 to 36.7°C. This temperature was around 10°C higher in comparison to the standard growth room conditions, i.e., 25±2°C temperature at a light intensity of 2,500 lux. High temperatures may lead to the alteration of cellular processes, such as protein expression (Koini et al., 2009; Stavang et al., 2009) and the hormonal regulation of development by, for example, cytokinin (Macková et al., 2013). The acquisition of thermotol- erance (acclimatization) requires the expression of heat shock proteins (HSPs) in order to avoid oxidative stress and involves the synthesis of abscisic acid (ABA) (Penfield, 2008). ABA is a well-known phyto- hormone, which inhibits the growth of plants under Duarte et al. ‐ Efficient and easy micropropagation of Morus nigra 437 environmental stress or pathogen attack (Sharp and LeNoble, 2002). Therefore, it could result in the reduced shoot and root formation in the nodal seg- ments of M. nigra under the extreme light and tem- perature in greenhouse conditions. Future experi- ments should use covers to reduce the light intensity and extreme temperatures in the flasks, similar to those used for the gerbera pre-acclimatization tests (Cardoso et al., 2013). The in vitro culture of micropropagated plantlets during elongation and rooting phase under green- house conditions is called as pre-acclimatization. This has been successfully executed in gerbera (Gerbera jamesonii) and was reported to promote the growth and acclimatization rate of gerbera plantlets and reduce the costs of micropropagation (Cardoso et al., 2013). The major differences between the previous and present studies lie within the partial control of environmental factors, which in the previous study were maintained as 25±5°C, PPFD 100 μmol m-2 s-1 and a 12-h photoperiod, i.e., natural growing condi- tions for gerbera. In contrast, in our study with M. nigra, the incidence of high light intensity (900-1800 μmol m-2 s-1), a temperature exceeding 30°C, and longer photoperiod (12:30 h) proved detrimental to in vitro as well as field-based development of plantlets. Growth room environment for 28‐days of in vitro cul‐ tivation provided optimum conditions for in vitro root‐ ing and acclimatization As shown in Table 2, the previously observed effect of in vitro conditions on the development of plantlets was also reflected during the acclimatization stage under greenhouse conditions. The nodal explants maintained in vitro under greenhouse condi- tions also resulted in reduced development of plantlets in the acclimatization stage. The duration of in vitro culture incubation also affected the development of acclimatized plantlets of M. nigra. Although a 7-day-incubation period result- ed in 96% survival, plantlets were better developed with a 28-day-incubation period before acclimatiza- tion (Table 2). The plants maintained for different durations under in vitro greenhouse conditions showed improvement only in leaf width (0.57 cm at 7-d versus 1.79 cm at 28-d), with no gain in terms of shoot length and number of leaves (Table 2; Fig. 2C). On the other hand, plants grown under culture room conditions showed better ex vitro acclimatization of M. nigra plantlets, as the shoots obtained after 28 days of in vitro incubation were longer (9.2 cm) and had greater leaf width (3.2 cm) compared to those incubated in vitro in the greenhouse (3.8 cm and 1.8 cm, respectively). Fig. 2 - Number of nodal segments with new shoots (A) and roots (B) along the period of 28-days of in vitro cultivation. Plants after 30 days from ex vitro acclimatization (C) and previous in vitro cultivated under growth room (left) and greenhouse (right). 438 Adv. Hort. Sci., 2019 33(3): 433-439 The culture room conditions of programmed and controlled temperature with low light intensity were conducive enough for the production of plantlets with healthy shoots and roots, and consequent suc- cessful acclimatization (100%) of Morus nigra. 4. Conclusions A single-step protocol using PGR-free culture medium for the in‐vitro shoot induction, multiplica- tion, and rooting was optimized for efficient large- scale production of M. nigra plantlets. The micro- propagated shoots of M. nigra were maintained in PGR-free culture medium for two years without any symptoms of morphological abnormalities or somaclonal variations. The in vitro rooting under cul- ture room conditions after 28 days presented better response during the acclimatization phase, with the production of plantlets of superior quality. 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