Middle East Journal of Applied Volume : 08 | Issue :04 |Oct.-Dec.| 2018 Sciences Pages: 1425-1436 ISSN 2077-4613

Micropropagation and ex vitro acclimatization of

Ahmed A. Barakat 1 and Mohamed K. Gaber2

1Botanical Garden Res. Dept., Hort. Res. Inst., Agric. Res. Center, Alexandria, Egypt. 2Plant Production Department, Faculty of Agriculture Saba Basha, Alexandria University, Alexandria, Egypt. Received: 25 Oct. 2018 / Accepted 10 Dec. 2018 / Publication date: 30 Dec. 2018

ABSTRACT Aglaonema commutatum is an ornamental foliage evergreen and important for interiorscaping due to its attractive foliage, easiness to grow and tolerance to low relative humidity and low light conditions and commonly referred to as ‘chinese evergreens’. It belongs to family , which commonly known as aroids and member of the aglaonema. The aim of this study is to develop a technique for micropropagation of A. commutatum plants using single nodal cuttings excised from soft cuttings cultured on full strength MS medium, 30g/l sucrose, 3g/l gelrite augmented with various concentrations of plant growth regulators as NAA, BA and their combinations. Different parameters including the mean number of shoots, shoot length (cm), mean number of leaflets, mean number of nodes and mean number of roots formed per propagule were studied during the time course of all tested stage. Neoformed shoots were divided into single nodes with one axillary bud per node each which were used for all micropropagation stages (i.e. initiation, multiplication and rooting). In general, the present study concluded that the best results for initiation stage was achieved, when MS medium were fortified with NAA and BA at 2.00 and 1.00 mg/l, respectively. Meanwhile, fortified medium with BA and NAA at 4.00 and 1.00 mg/l, consecutively, gave rise to the best results for multiplication stage. Regarding rhizogenesis stage, the best results were recorded when the neoformed shoots of multiplication stage were divided singly and cultured on MS medium plus IBA and NAA at 0.50 and 0.25 mg/l, respectively which led to the highest mean number of roots formed per propagule. Neoformed plantlets were acclimatized ex vitro and in vivo vigorously in mixture of perlite and peatmoss at (1:1) or (1: 2) or (1: 4) or (2:2) and (2:4) consecutively; which resulted in the highest mean value of survival percentage/plant (100%) and showed true-to-type plants ex vitro.

Keywords: Aglaonema commutatum, Araceae, micropropagation, in vivo, in vitro, ex vitro, acclimatization, BA, NAA.

Introduction Aglaonema commutatum Schott, commonly referred to as ‘chinese evergreens’, is a monocotyledonous genus belonging to the family Araceae, which commonly known as aroids, it is have more than three thousand species that are mostly herbaceous either as terrestrial, aquatic, or epiphytic (Mayo et al., 1997; Bown, 2000). The genus Aglaonema is comprised of 21 species which inhabit humid and heavily shaded forests of many territories of Asia (Chen et al., 2003; Govaerts and Frodin, 2002). Most Aglaonema species are open-pollinated; a few may exhibit apomixis, such as A. costatum ‘Foxii’. Propagation of aglaonema is by either seeds or vegetative tissues, (mainly by tip cuttings or division). The basic number of chromosomes could be x = 6, with subsequent polyploidy in many cases (Jones, 1957). Ornamental aroids are a basic foliage plant account and commerce for ornamental foliage plant sales in the United States (Henny et al., 2004). Almost all aglaonema are hybrids developed through traditional breeding. (Henny et al., 2003). Aglaonema has been produced as a foliage due to its attractive foliage, easiness to grow and tolerance to low light conditions and low relative humidity (Henny, 2000; Chen et al., 2002). Nevertheless, the rooting of its cuttings and division of basal shoots are the basic methods of propagation since non-simultaneous flowering and short life span of the pollen make sexual reproduction difficult. Micropropagation techniques are advanced vegetative propagation technique for producing a large amount of uniform and pathogen-free transplants in a short period of time and limited space

Corresponding Author: Mohamed K. Gaber, Plant Production Department, Faculty of Agriculture Saba Basha, Alexandria University, Alexandria, Egypt. E-mail: [email protected] 1425 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613

(Razdan, 2003; George et al., 2008), decreases greenhouse space needed for stock plant production and provides growers with lines of tissue-cultured plantlets grown in cell plug trays on a year-round basis (Chen and Henny, 2008). Adoption of in vitro propagation has reduced the time-spanned for plant introduction and new release. Using micropropagation techniques, a new cultivar can be improved quickly enough to reach commercial production levels (Henny and Chen, 2003). Nevertheless, it has not been principally effective mainly due to the difficulty of establishing sterile culture (Chen and Yeh, 2007), low rate of shoot multiplication (Zhang et al., 2004), and lack of detailed methodological information on the in vitro propagation technique (Mariani et al., 2011). The previous studies showed that average in vitro shoot number depends on the cultivar and protocol (Chen and Yeh, 2007; Fang et al., 2013; Mariani et al., 2011). For instance, in vitro shoots were formed every four weeks from stem nodes of aglaonema on a medium fortified with 2.5 mg/l BA and 0.05–0.1 mg/l NAA (Zhang et al., 2004), aglaonema ‘White Tip,’ using stem nodal sections with 6.8 mg/l BA after 60 day of culture (Chen and Yeh, 2007), Another report by (Yeh et al., 2007) induced direct shooting on ‘White Tip’ explants using 1.1–2.2 mg/l dicamba and 2.2 mg/l (TDZ). Shoots were formed from the rhizome explants cultured on medium supplemented with 3 mg/l BA after eight weeks, but it was reported for an aquatic species (Ahmad et al., 2008). In aglaonema ‘Cochin,’ direct organized shoots were derived from shoot explants cultured on medium supplemented with 1.5 mg/l TDZ and 3 mg/l BA at the 5th subculture, but the shoot prefoliation rate was decreased and no technical details on rooting was provided (Mariani et al., 2011). Culture media supplemented with cytokinins are crucial for shoot multiplication in aroids (Hussein, 2004). Therefore, the aim of the present study is to develop a reliable and efficient protocol for micropropagation and acclimatization of aglaonema plantlets. Materials and Methods

The experiments regarding the effect of different concentrations of certain growth regulators and their combinations on micropropagation of A. commutatum plantlets using nodal segments as explants were conducted in the Plant Tissue Culture Laboratory of The Faculty of Agriculture Saba Basha, Alexandria University, during the period of 2016 and 2017.

Plant materials The explant materials were picked up from healthy mother plants grown in the greenhouse of Antoniades Botanical Garden, Horticultural Res. Inst., Agric. Res. Center, Alexandria, Egypt. The collected material, were brought to the laboratory to cooled process, old leaves were removed and washed after that to be ready for sterilization and tissue culture manipulation, then shoot explants from cuttings were washed thoroughly in the water, using liquid soap for 30 min., then the excised explants were placed under running tap water for 90 minutes then dipped in 70% ethanol for 15 sec. After pretreatment with ethanol, the explants were rinsed with double distilled water twice, to lower the toxic effect of ethanol. Nodal segments of only (1cm) long nodal segment which contained a single node were then surface sterilized with concentration of mercuric chloride (HgCl2) at 0.1% (v/v) with a few drops of wetting agent “Tween-20” (surfactant agent) for fifteen minutes. The similar procedure was repeated, but the explants were immersed in concentration of sodium hypocholrite solution (NaOCl) at 30%. After the surface sterilization of explants with mercuric chloride and sodium hypocholrite solution were decanted and the explants were rinsed with sterile double distilled water for four times, so as to lower the toxic effects of HgCl2 and NaOCl and became ready for culturing.

In vitro experimental stages

1. Initiation stage, during this stage explants were cultured on solidified Murashige and Skoog medium (1962) solidified with gelrite (3g/l). The pH of the tested media was adjusted to 5.7 before adding gelrite, and then sterilized autoclaving at 121°C for 20 min., then explants were cultured into the given MS medium which contained different concentrations of cytokinin (BA at three concentrations: 0.00 (nil), 0.50, and 1.00 mg/l, in combinations with auxin (NAA) at four concentrations 0.00 (nil), 1.00, 2.00, and 4.00 mg/l.

1426 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613

2. Multiplication stage, in this the neoformed propagules of the initiation stage were sectioned into single leaflets nodes (ca. 1 cm). The excised nodal cuttings explants of the different positions were cultured, randomly, on multiplication media which supplemented with BA at four concentrations: 0.00 (nil), 2.00, 4.00, and 8.00 mg/l, in combinations with NAA at three concentrations: 0.0 (nil), 1.00, and 2.00 mg/l. 3. Rhizogenesis The obtained shoots from the multiplication stage were, individually, cultured on a rooting medium, contained two types of auxins were tested, Indole-3-Bytric acid (IBA) at four concentrations: 0.00 (nil), 0. 25, 0.50 and 1.00 mg/l, in combinations with NAA at three concentrations: 0.0 (nil), 0. 25 and 0.50 mg/l. Generally, each treatment was represented by 10 jars and three explants per each jar (175 ml) containing 20 ml medium. The culture jars and the tested media were solidified and autoclaved as mentioned earlier. The explants were cultured on the sterilized media, vertically, and incubated in growth chamber at 25 ± 1°C temperature under 16 hr daily light and 8 hr darkness illumination by a florescent light intensity of 2880 Lux (40µmol m-2s-1 PPF). After 8 weeks of culture, the data were collected. Generally, the following characters were recorded per propagule at initiation, multiplication and rooting stages after 8 weeks in culture; mean number of shoots formed per propagule, mean shoot length (cm) per propagule, mean number of leaflets formed per propagule, mean number of nods formed per propagule, mean number of roots formed per propagule. Acclimatization of neoformed plantlets, the plantlets produced from rooting stage were washed out of solidified medium under running tap water, followed by immersing them into fungicide for 25 sec. They were, then, transplanted ex vitro in small plastic pots (10 cm) plastic pots contained a combination of an autoclaved mixture of the perlite (0, 1, 2 volume) and peatmoss at (0, 1, 2, 4 volume) each; and one constant volume of washed and autoclaved sand. Then, they were arranged in a factorial experiment and finally placed in transparent plastic bags (ex vitro), to maintain high relative humidity at (RH) 85% and 27±1°C, for hardening-off. However, the tested pots with different media were rearranged, randomly, weekly within same plot to devoid the experimental error. Ten days later, the plastic bags were perforated for gaseous exchange, then transferred into plastic house (in vivo) and continued for further hardening. After four weeks, the plastic bags were removed and the acclimatized plantlets were watered, as needed, and fertilized weekly with mineral fertilization (20:20:20) at 1g/l. Concerning the acclimatization stage, the following traits were determined after four weeks in culture; average survival percentage (%) per plant, average number of neoformed shoots per plant, average plant height (cm) per plant, average number of neoformed leaflets per plant.

Experimental design and statistical analysis, all the experiments carried out during this study were designed as factorial experiments layout in completely randomized design (Gomez and Gomez, 1984). Recorded data were analyzed statistically using analysis of variance technique (ANOVA) and means were compared by Duncan’s multiple range tests (Steel et al., 1997) and significance was determined at p≤ 0.05.

Results and Discussion

The collected and analyzed results will be presented and discussed as follows:

1. Initiation stage Results presented in Table (1) and Figure (1) exhibit that both applied growth regulators (BA and NAA) and their combinations exerted significant effects on the initiation stage’s characters of single node explants of A. commutatum grown in vitro. Concerning the main effect of BA on the studied characters, i.e. numbers of shoot, shoot length, number of leaflets, roots formed per propagule showed that BA levels had a significant effect on the given traits. However, the highest level of BA (1.00 mg/l); gave rise to the highest mean value of number of shoots per propagule (1.86). This finding may be attributed to cytokinin efficiency in stimulation cell division and morphogenesis (shoot initiation/bud formation) in tissue culture and break of apical dominance and release growth of lateral buds (Stern et al., 2004; George et al., 2008). Regarding the main effect of NAA tested levels on the above-mentioned traits; NAA levels had a

1427 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613 significant effect on the given characters. However, the highest mean values were always recorded when culture medium was supplemented with 2.00 mg/l NAA for number of shoots, shoots length, number of leaflets and number of roots formed per propagule. The above-mentioned results indicated, generally, that increasing the mean values of the studied characters was concomitant with increasing BA, these results could be attributed to the mode of action of cytokinins on stimulation of both cell division and promotion growth of axillary shoots in plant tissue culture (Trigiano and Gray, 2005; George et al., 2008). Many reports demonstrated that BA is superior to other cytokinins for the release of axillary buds from apical dominance in other Araceae members including Dieffenbachia (Elmahrouk et al., 2006) and Spathiphyllum (Dewir et al., 2006). When cytokinins were used with auxin the number of shoots per explant increased in comparison with treatments with BA alone. Meanwhile, Fang et al. (2013) reported that no adventitious shoot formation was observed on Aglaonema ‘Lady Valentine’ stem nodal segments when BA was the sole PGR in the medium.

Table 1: Effect of different levels of NAA and BA (mg/l) and their combinations on the initiation stage of Aglaonema commutation cultured in vitro for 8 weeks. Characters BA NAA levels (mg/l) Mean Significance levels BA BAΧNAA 0.00 1.00 2.00 4.00 NAA BA (mg/l) (a) Mean number of shoots formed/propagule: 0.00 0.44 0.67 1.22 0.78 0.78 ** ** ** 0.50 1.11 1.33 1.44 1.22 1.28 1.00 1.11 1.44 3.22 1.67 1.86 Mean (NAA) 0.89 1.15 1.96 1.22 L.S.D. (0.05) 0.32 0.28 0.56 (b) Mean shoot length (cm)/propagule: 0.00 0.29 0.79 1.66 1.01 0.94 ** ** ns 0.50 1.86 2.42 2.83 1.93 2.26 1.00 2.16 2.87 3.89 2.66 2.89 Mean (NAA) 1.43 2.03 2.79 1.87 L.S.D. (0.05) 0.28 0.24 0.49 (c) Mean number of leaflets formed/propagule: 0.00 0.89 1.11 1.89 1.22 1.28 ** ** ns 0.50 2.11 2.44 3.22 2.89 2.67 1.00 3.22 3.44 4.89 3.67 3.81 Mean (NAA) 2.07 2.33 3.33 2.59 L.S.D. (0.05) 0.51 0.44 0.89 (d) Mean number of nodes formed/propagule: 0.00 1.11 1.56 2.44 1.56 1.67 ** ** ns 0.50 2.78 3.11 4.00 3.78 3.42 1.00 4.11 4.11 6.00 4.56 4.69 Mean (NAA) 2.67 2.93 4.15 3.30 L.S.D. (0.05) 0.59 0.51 1.02 (e) Mean number of roots formed/propagule: 0.00 0.00 0.00 0.33 0.00 0.08 ** ns ns 0.50 0.00 0.33 0.56 0.22 0.28 1.00 0.00 0.00 0.78 0.11 0.22 Mean (NAA) 0.00 0.11 0.56 0.11 L.S.D. (0.05) 0.21 0.18 0.36 L.S.D. (0.05) = Least significant difference test at 0.05 level of probability. *, **: Significant or highly significant, ns: not significant

Further the mode of action of auxin (NAA) also exerts profound effect either endogenously or exogenously on cultured tissues, and is capable of controlling various distinctive processes such as cell growth and elongation (Trigiano and Gray, 2005). Furthermore, it is known its role and mode of action for their abilities to enhance root formation was reported by many research workers (George et

1428 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613 al., 2008; Gaber, 2012 Douban, 2016). Concerns to the interaction between both applied growth regulators at either 0.50 or 1.00 mg/l BA, and 1.00 and 2.00 mg/l NAA; expressed significant effects on the same tested traits. For example, Elmahrouk et al., (2016) ascertained that the effectiveness of combining NAA with cytokinins on increasing shoot multiplication of Aglaonema ‘Valentine’. Similar findings on A. commutatum were reported by Zhang et al., (2004) and Fang et al., (2013). The proportion of auxin–cytokinin combinations is a determinant factor for stem formation and the hormone balance has been established between growth regulators and determines the type of buds induced (George, 1993). The inherent endogenous auxin and cytokinin levels must have also played part in bud differentiation (Pierik, 1987). These results suggest that one mode of action for auxins could be to down regulate both local cytokinin synthesis and cytokinin export from medium; this might influence the endogenous cytokinin levels and lead to the activation of buds (Sato and Mori, 2001).

Fig. 1: Initiation stage of A. commutatum axillary buds grown in vitro on MS medium augmented with BA at 1.00 mg/l and NAA at 2.00 mg/l over 60 days.

2. Multiplication stage Results in Table (2) and Figure (2) describe the effect of various levels of both growth regulators and their combinations on the studied characters of A. commutatum. Respecting the main effect of BA, it showed that its presence in the culture medium at 4.00 mg/l; resulted in the highest mean number of shoots, number of leaflets and number of nodes per propagule. Meanwhile, its absence from the basal medium; led to the highest mean of shoot length and roots formed per propagule. On the other end, the main effect of NAA declared that augmenting the basal medium with it at 1.00 mg/l; brought about the highest number of shoots, shoot length, number of leaflets and number of roots per propagule. Meanwhile, the interaction between BA and NAA at 4.00 and 1.00 mg/l, respectively, led to the highest mean number of shoots, shoot length and number of leaflets formed per propagule. It could be inferred from above results that BA at 4.00 mg/l, was the optimal concentration for better performance of this particular hormone. Whereas, any higher or lower deviation from this concentration; the propagules showed poor performance. These results could be taken place due to the cytokinins mode of action on stimulation both cell division and promotion growth of axillary shoots in plant tissue culture as, also, found by Trigiano and Gray, (2005) and George et al., (2008). The superiority of BA over other cytokinins such as kinetin (Kin), 2iP and TDZ in promoting shoot elongation has also been reported in other ornamental aroids such as Spathiphyllum cannifolium (Dewir et al., 2006), Caladium bicolor (Ali et al., 2007), Zantedeschia aethiopica (Kozak and Stelmaszczuk 2009), and Diffenbachia compacta (Azza et al., 2010). These results are in parallel with Elmahrouk et al., (2016) who found that the highest shoot proliferation of aglaonema ‘Valentine’ (2.5) was obtained on MS medium supplemented with 3 mg/l BA and 1 mg/l NAA. Chen and Yeh, (2007) found that the shoots were formed on each stem nodal section on medium supplemented with 6.8 mg/l BA after 60 day of culture. Increased shoot numbers with BA have been reported for Anthurium andreanum Lind. (Kunisaki, 1980), and Spathiphyllum floribundum; (Ramirez-Malagon et al., 2001). Increasing BA concentration up to 7.5 mg/l BA; resulted in maximum shoot proliferation of A. simplex (Laohavisuti and Mitrnoi, 2005). In addition, Dewir et al., (2006) reported that the largest number of shoots of Spathiphyllum cannifolium was obtained with BA and IBA. Furthermore, BA considers as antagonist for rhizogenesis and in favour of stimulates cell division, stimulates morphogenesis (shoot initiation/bud

1429 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613 formation) in tissue culture and stimulates the growth of lateral buds-release of apical dominance. (Mauseth, 1991; Davies, 1995).

Table 2: Effect of different levels of BA and NAA (mg/l) and their combinations on the multiplication stage of Aglaonema commutation cultured in vitro for 8 weeks. NAA BA levels (mg/l) Mean Significance Characters levels NAA BAΧNAA 0.00 2.00 4.00 8.00 BA NAA (mg/l)

(a) Mean number of shoots formed/propagule: 0.00 0.78 1.44 2.78 1.89 1.72 ** ** **

1.00 1.33 3.67 6.56 4.33 3.97

2.00 1.33 3.00 4.33 2.78 2.86

Mean (BA) 1.15 2.70 4.56 3.00

L.S.D. (0.05) 0.38 0.33 0.66 (b) Mean shoot length (cm)/propagule: 0.00 3.40 3.01 2.11 0.72 2.31 ** ** **

1.00 5.82 3.60 3.32 1.93 3.67

2.00 3.70 3.19 2.91 2.48 3.07

Mean (BA) 4.31 3.27 2.78 1.71

L.S.D. (0.05) 0.35 0.31 0.61 (c) Mean number of leaflets formed/propagule: 0.00 1.33 2.78 3.67 3.00 2.69 ** ** **

1.00 2.11 3.22 6.00 4.67 4.00

2.00 3.00 3.56 5.22 3.67 3.86

Mean (BA) 2.15 3.19 4.96 3.78

L.S.D. (0.05) 0.46 0.40 0.80 (d) Mean number of nodes formed/propagule: 0.00 2.11 3.78 4.67 3.89 3.61 ** ** **

1.00 3.22 4.11 6.78 5.44 4.89

2.00 4.22 4.67 6.00 4.44 4.83

Mean (BA) 3.19 4.19 5.81 4.59

L.S.D. (0.05) 0.51 0.44 0.88 (e) Mean number of roots formed/propagule: 0.00 0.00 0.00 0.00 0.00 0.00 ** ** **

1.00 1.11 0.78 0.22 0.00 0.53

2.00 1.00 0.00 0.00 0.00 0.25

Mean (BA) 0.70 0.26 0.07 0.00

L.S.D. (0.05) 0.16 0.19 0.33 L.S.D. (0.05) = Least significant difference test at 0.05 level of probability. *, **: Significant or highly significant, ns: not significant

However, it is known that average number of leaves per shoot and number of nodes is one of the important growth factors and is in directly proportional relationship to the length of the shoot, and if the shoot length increases, the number of leaves and number of nodes increase as well (Waseem et al., 2009). Gaber, (2012) found that the main effect of NAA declared that augmenting the basal medium with it at 0.250 mg/l, brought about the highest shoot length. However, its presence in the basal medium at 1.00 mg/l, led to the highest mean number of leaflets, nodes and roots formed per propagule. Also, Elmahrouk et al., (2006) found that NAA promoted the shoot growth elongation which has more internodes and leaves of Dieffenbachia sp. regarding the mean number of roots formed /propagule, the presence of NAA at 0.75 mg/l or absence of BA brought about the highest rhizogenesis. This finding might be taken place due to the well-known role of auxin in inducing of root formation (George et al., 2008). The use of a cytokinin in combination with an auxin can be effective for increasing shoot multiplication (Dewir et al., 2006; Shen et al., 2007). With respect to

1430 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613 the combinations between both growth regulators between BA and NAA led to significant effects on the studied traits.

Figure (2). Multiplication stage of A. commutatum explants grown in vitro on MS medium augmented with BA at 4.00 mg/l and NAA at 2.00 mg/l over 60 days.

3. Rhizogenesis stage: Results in Table (3) and figure (3) showed that the applied both auxin levels exerted significant effects on the studied characters of A. commutatum. For IBA as a main effect, the mean number of shoots, it was noticed that IBA at 0.50 mg/l; led to the highest mean values, and the main effect of NAA indicated that 0.25 mg/l of NAA; recorded the highest mean value of the mean number of shoots. While, the interaction between IBA at 0.50 and NAA at 0.25 mg/l; caused the highest mean value of the studied trait. Concerning, the main effect of IBA tested levels on the mean number of shoot length; there was highly significant effects of the given trait at 0.50 mg/l. On the other side, the main effect of NAA at 0.25 mg/l recorded the especially highest mean value of the mean number of shoot length. The interaction between IBA and NAA recorded no highly significant effects of the given trait at 0.5 mg/l IBA and 0.25 mg/l NAA. Concerns the number of leaflets formed per propagule, the effect of IBA, exerted highly significant effect on the given trait at 0.50 mg/l. In case of the main effect of NAA it had highly significant effect on the on the given trait at 0.25 mg/l. The interaction between both BA and NAA at 0.50 and 0.25 mg/l, respectively, resulted in the highest mean values. Regarding the number of roots formed per propagule, results showed that there is a proportional relationship between IBA and the given trait the highest mean value recorded at 0.50 mg/l (4.63). On the other side, the main effect of NAA indicated that supplying MS-basal medium with NAA at 0.50 mg/l achieved the highest mean value of the mean number of roots (3.92). The interaction between IBA at 0.50 mg/l and NAA at 0.25 mg/l; respectively, gave rise to the highest mean value of the given trait (6.11). The obtained results showed that the used auxins (NAA and IBA), in general, produced the best results in almost all studied traits. These results could be explained on the basis that auxin induced number of responses involved cell division, cell enlargement, protein and nucleic acids synthesis which are concomitants of auxin-induced growth and changes in wall plasticity of plant cell and increase the apical dominance as there are essential and rapid processes involved in growth and elongation. Several studies have described IBA as a suitable auxin for adventitious root induction and it was often found to be superior to IAA and NAA because of its more stable nature. (Jahan et al., 2009) induced roots from anthurium shoots after six weeks of culturing on medium containing 1.0 mg/l IBA. Moreover, Atak and Celik, (2009) obtained a rooting rate of 95-98% for two Anthurium andreanum cultivars when 1 mg/l IBA was included in the medium. In aglaonema ‘Cochin,’ shoots rooted and developed on medium containing 3.0 mg/l IBA (Mariani et al., 2011). Fang et al., (2013) reported that roots were successfully induced supplementing medium with 0.5-2 mg/l IBA under both in vitro and ex vitro conditions.

1431 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613

Table 3: Effect of different levels of IBA and NAA (mg/l) and their combinations on rhizogenesis stage of Aglaonema commutation cultured in vitro for 8 weeks. NAA IBA levels (mg/l) Mean Significance Characters levels NAA IBAΧNAA 0.00 0.25 0.50 1.00 IBA NAA (mg/l) (a) Mean number of shoots formed/propagule: 0.00 1.00 1.22 1.56 1.89 1.42 ** ** * 0.25 1.33 1.78 2.67 1.78 1.89

0.50 1.11 1.11 1.78 1.33 1.33

Mean (IBA) 1.15 1.37 2.00 1.67 L.S.D. (0.05) 0.30 0.26 0.51 (b) Mean shoot length (cm)/propagule: 0.00 1.64 2.28 2.78 2.41 2.28 ** ** ns 0.25 2.46 3.07 3.37 3.21 3.03

0.50 2.67 3.24 3.64 3.36 3.23

Mean (IBA) 2.26 2.86 3.26 2.99 L.S.D. (0.05) 0.25 0.22 0.43 (c) Mean number of leaflets formed/propagule: 0.00 2.44 3.78 4.33 5.00 3.89 ** ** ns 0.25 3.00 4.44 5.33 5.00 4.44

0.50 3.56 4.78 5.22 4.67 4.56 Mean (IBA) 3.00 4.33 4.96 4.89 L.S.D. (0.05) 0.52 0.45 0.90 (d) Mean number of nodes formed/propagule: 0.00 3.67 5.00 5.22 6.00 4.97 * ** ns 0.25 4.00 5.44 6.22 5.89 5.39

0.50 4.56 5.67 6.11 5.56 5.47 Mean (IBA) 4.07 5.37 5.85 5.81 L.S.D. (0.05) 0.41 0.48 0.83 (e) Mean number of roots formed/propagule: 0.00 0.33 1.11 2.67 1.78 1.47 ** ** ** 0.25 1.67 3.33 6.11 4.00 3.78

0.50 2.22 4.00 5.11 4.33 3.92

1.41 2.81 4.63 3.37 3.37 Mean (IBA) L.S.D. (0.05) 0.48 0.41 0.83 L.S.D. (0.05) = Least significant difference test at 0.05 level of probability. *, **: Significant or highly significant, ns: not significant.

Fig. 3. Rooting stage of A. commutatum shoots grown in vitro on MS medium plus IBA at 0.50 mg/l and NAA at 0.25 mg/l over 60 days.

Chen and Yeh, (2007) showed that all microcuttings rooted and produced maximum root numbers and lengths with auxins. Waseem et al., (2008) reported that IBA showed its superiority over

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all other tested Auxins, when used alone. Our results were further confirmed by the previous findings of Munshi et al., (2004); Awal et al., (2005); Rajani and Patil, (2009). Regarding the interaction between both applied growth regulators between IBA at 0.50 mg/l and NAA at 0.25 mg/l, led to significant effects on the almost of vegetative growth. These results were in agreement with Mariani et al., (2011) on aglaonema plants.

Ex vitro and in vivo acclimatization Results presented in Table (4) exhibit that both applied mixtures of perlite and peatmoss (v/v) and their combinations, in addition to fixed volume (1 portion) of sand, on acclimatization of neoformed plantlets of A. commutatum grown ex vitro for four weeks and as shown in Figure (4). Concerning the average of survival percentage/ plant, perlite had a highly significant effect on this trait. The highest mean value was recorded at levels (1v/v) [80.56%]. Also, peatmoss had highly significant effect on the graven traits was recorded at levels (2v/v) [88.89%]. Meanwhile, the interaction between peatmoss and perlite exerted highly significant effect on the given traits. However, the combination of perlite and peatmoss at either (1:1) or (1: 2) or (1: 4) or (2:2) and (2:4), respectively, resulted in the highest mean value of survival (100%).

Table 4: The effect of different potting mixtures of perlite and peatmoss (v/v) and their combination on the acclimatization of neoformed plantlets of Aglaonema commutation after four weeks ex vitro. Perlite Peat moss levels Mean Significance Peatmoss Characters levels Perlite 0.0 1.0 2.0 4.0 Peatmoss Perlite Χ Perlite (a) Average survival percentage (%) / plant: 0.0 0.00 55.56 66.67 44.44 41.67 ** ** * 1.0 22.22 100.00 100.00 100.00 80.56

2.0 11.11 44.44 100.00 100.00 63.89

Mean Peat moss 11.11 66.67 88.89 81.48 L.S.D. (0.05) 20.00 17.00 35.00 (b) Average number of neoformed shoots / plant: 0.0 0.00 0.67 0.67 0.67 0.50 ** ** * 1.0 0.22 1.00 1.67 1.00 0.97

2.0 0.22 0.44 1.00 1.00 0.67

Mean Peat moss 0.15 0.70 1.11 0.89 L.S.D. (0.05) 0.24 0.21 0.42 (c) Average plant height (cm) / plant: 0.0 0.00 8.12 8.73 8.22 6.27 ** ** ** 1.0 0.91 12.66 17.97 14.87 11.60

2.0 0.69 5.49 13.04 16.67 8.97 Mean Peat moss 0.53 8.76 13.25 13.25 L.S.D. (0.05) 2.39 2.07 4.14 (d) Average number of neoformed leaves/plant: 0.0 0.00 3.44 3.78 3.33 2.64 ** ** ns 1.0 1.33 6.67 7.78 6.11 5.47

2.0 1.22 2.56 5.22 6.00 3.75 Mean Peat moss 0.85 4.22 5.59 5.15 L.S.D. (0.05) 1.05 1.22 2.12 L.S.D. (0.05) = Least significant difference test at 0.05 level of probability. *, **: Significant or highly significant, ns: not significant

Respecting the average number of neoformed shoots / plant, perlite and peatmoss and their interactions, exerted significant effects on the given traits. In case of perlite, the main effect at (1 v/v), brought about the highest mean value. On the other side, peatmoss had highly significant effect on number of neoformed shoots / plant at (2 v/v). However, the interaction between both added levels of

1433 Middle East J. Appl. Sci., 8(4): 1425-1436, 2018 ISSN 2077-4613 perlite and peatmoss at (1:2) respectively, resulted in the highest mean values. In this respect, a material substance as peatmoss is one of the most important constituents of media due to its capacity in affecting plant growth either indirectly or directly. Indirectly, improves the physical conditions of media by enhancing aggregation, aeration (8%) and water retention (77%), thereby creating a suitable environment for root growth (Sensi and Loffredo, 1999). On the other hand, perlite is known to have a moderate capacity to retain water (38%) and provides aeration (25%) and its neutral pH and the fact that it is sterile and weed–free. Hence, it is ideal for use as container growing substratum (Abido, 2016). Also, it is known that perlite decreases the bulk density of the soils and increases the porosity. In addition, Li et al., (2012) found that some of the plantlets were removed from rooting media, washed, and then transferred to pots containing a mixture of sterile soil, peat and vermiculite in a ratio of 1:1:1. Newly potted plantlets were covered with polythene bags for 1 week before transferring to a research greenhouse. Survival percentage was recorded four weeks after transplanting and acclimatized plants with 100% success. On brief, this study developed a reliable and efficient protocol for in vitro propagation in complete cycle, successfully.

Fig. 4: Acclimatization stage of A. commutatum plantlets grown on rooting medium of peatmoss and perlite at (2:1, v/v) over 30 days.

References

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