In vitro Propagation and Ex vitro Acclimatization of Solidago Canadensis Using Nodal Segment Explants

INTRODUCTION

Solidago canadensis, L.or goldenrod is a rhizomatous perennial (Weber and Jakobs, 2005) which belongs to the family Asteraceae. The genus name derived from the Latin solidus meaning whole, refering to its traditional healing properties.

Solidago canadensis are produced annually by cuttings from a perennial branched rhizome system. The species is self-incompatible, self fertile (Schmid and Dolt, 1994) and diploid 2n = 2X = 18 (Melville and Morton, 1982). To maintain its quality, crop should be replanted every 2-3 years (Nowak and Rudnicki, 1990), or as early as 1/4 of the flowers have opened (Bartels, 2001). It became one of the most important commercial cut flowers in the recent five years. With respect to landscape importance, the plant is used in meadow and natural gardens, borders, cut flower, bee and butterfly gardens because it provides good colour and contrast for the late summer to early fall (LiPing et al, 2003). Some investigators are using Solidago plants as a source of biochemical secondary metabolites. The extract of Solidago plants inhibit  the growth of human gastric adenocarcinoma, spasmolytic, antihypertensive and diuretic effects at the same time, the extract of Solidago plants is very important in immunodularty and anti-inflamatory activity (Kruedener et al. ,1995; Matsunaga et al., 1999; Ivan-Razmilic et al., 2000; Sampson et al., 2000).Roots used in treatment of burns.Tea from flowers used to treat fevers and snakebites. Crushed flowers can be chewed for sore throats. Leaf extracts are diuretic.  Therefore, attention about this genus is in upscalling for more production.

 Pertaining in vitro propagation of S. canadensis, Abd-Elrahman (2002) reported that the best result for surface sterilization was done by using 40% clorox concentration(v/v) [NaOC,l 2.0%] produced the highest percentage of the free - contamined explants (80%) on."Toto"cv. and (85%) on "Tara" cv. and increasing strength of medium; produced the greatest shootlet number per explants for "Toto" and "Tara" cvs Li et al. (2012) found that the nodal explants with axillary buds of Solidago canadensis L. were collected from field-grown plants of goldenrod. Also, Li et al. (2012) found that the nodal explants with axillary buds of Solidago canadensis L. were collected from field-grown plants of goldenrod. The excised nodal seg­ments (0.5-1 cm long) without leaves were surface sterilized in 70% (v/v) ethanol for 30 to 40 s, followed with 8-min in 1 g/l HgCl₂, and rinsed five times with autoclaved dis­tilled water. Further, Paul et al. (2013) stated that various explants (node, internode and leaves) of Solidago virgaurea L. were thoroughly washed in running tap water for 30min. followed by the treatment in 1% Labolene, (a neutral detergent Qualigens, India) for 3min. and finally rinsed with the distilled water for 4-5 times to remove the surface microflora. The washed explants were surface sterilized with 0.1% (w/v) aqueous mercuric chloride (HgCl₂) for 3 min. and the chemical sterilent was removed by rinsing the materials with sterilized and cooled distilled water 4-5 times. However, Ailstock (1985) reported that explants obtained from terminal and subterminal nodes of vegetative stems were cultured on various media combinations. Adventitious shoots were initiated within three weeks on Murashige’s minimal organic medium (Murashige and skoog,1962) supplemented with 1-3 mg/l Kinetin. These shoots, when separated and placed on similar media with and without auxins, formed roots within five days and could be transferred to peat pots for greenhouse culture in 10 days. However, due to the greatest importantce of this plant species economically, the present study was undertaken to develop an integrated protocol for in vitro and ex vitro acclimatization of Solidago canadensis.

MATERIALS AND METHODS

The experiments regarding the effect of different concentrations of certain growth regulators and their combinations on micropropagation of Solidago canadensis 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 2012 to 2015.

Plant materials:

The explant materials were collected from 3-months old Solidago canadesis, L., kindly, gifted by a commercial farm (3H Co.) on Cairo-Ismailia highway. The healthy mother plants were grown under plastic house conditions. The collected material, were brought to the laboratory to process the washing and to be ready for sterilization and tissue culture manipulation then the shoots used from cuttings were washed thoroughly in the water, using liquid soap for 30 min. The excised explants were placed under running tap water for 30 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 different concentrations of mercuric chloride (HgCl₂) at 0.05, 0.1, 0.15 and 0.2% (v/v) with a few drops of wetting agent “Tween-20” (surfactant agent) for five minutes (Ilahi et al., 2007). The similar procedure was repeated, but the explants were immersed in different concentrations of sodium hypocholrite solution (NaOCl) at 30, 35, 40 and 45% (v/v).  After the surface sterilization of explants with mercuric chloride and sodium hypocholrite solutions 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. Determination of contamination ratio for each sterilant agent used in sterilization of culture explants was done.

In vitro experimental stages

1. Initiation stage

Explants were cultured on solidified Murashige and Skoog (1962) medium which 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 four concentrations: 0.0 (nil), 0.25, 0.50 and 0.75 mg/l, in combinations with auxin (NAA) at five concentrations 0.0 (nil), 0.5, 1.0, 1.5 and 2.0 mg/l.

2. Multiplication stage

The neoformed propagules of the initiation stage were sectioned into single leaflets nodes. The excised nodal cuttings explants of the different positions were cultured, randomly, in the multiplication media which supplemented with BA at five concentrations: 0.0 (nil), 0.5, 1.0, 1.5 and 2.0 mg/l, in combinations with NAA at five concentrations: 0.0 (nil), 0.25, 0.50, 0.75 and 1.0 mg/l.

3. Rhizogenesis

The obtained shoots of Solidago canadesis from the multiplication stages were, individually, cultured on a rooting medium, contained MS salts, sucrose at 30 g/l. and two types of auxins were tested, IBA at five concentrations: 0.0 (nil), 0.5, 1.0, 1.5 and 2.0 mg/l, in combinations with NAA at five concentrations: 0.0 (nil), 0.5, 1.0, 1.5 and 2.0 mg/l.

Generally, each treatment was represented by 3 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 under 16 hr daily light and 8 hr darkness illumination by a florescent light intensity of 2880 Lux (40µmol m^-2s^-1 PPF).

Acclimatization of neoformed plantlets

The plantlets produced from rooting stage of Solidago canadensis was washed out of solidified medium under running tap water, followed by immersing them into Rizolex-T50 WP (1g/l) [From Sumitomo Chemical Co. Ltd., Osaka, Japan] fungicide for 25 sec. They were, then, transplanted ex vitro in small plastic pots (10cm) plastic pots contained an autoclaved mixture of the perlite and peatmoss at (0, 1, 2, 3, volume) each; and one constant volume of washed and autoclaved sand. The perlite has a bulk density of about (0.03- 0.150 g/cm³) and porosity about 95%, while the peatmoss has a bulk density of about (0.250 g/cm³) and porosity about (95- 98%). Then, they were arranged in a factorial experiment and finally placed in transparent plastic bags (ex vitro), to maintain high relative humidity at (RH) 80% and 28±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 three weeks, the plastic bags were removed and the acclimatized plantlets were watered, as needed and fertilized, weekly, with N: P₂O₅: K₂O (20:20:20) equivalent to1g/l (AGRO 4).Generally, the following characters were recorded per propagule at initiation, multiplication and rooting stages for both tested cultivars after four weeks in culture.

Concerning the acclimatization stage, the following traits were determined:

1. Average survival percentage (%) / plant.

2. Average number of neoformed shoots / plant

3. Average plant height (cm) / plant

4. Average number of neoformed leaflets/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 L.S.D (Steel et al., 1997) and significance was determined at p≤ 0.05.

Generally, the following characters were recorded per propagule at initiation, multiplication and rooting stages after 35 days in culture:

1. Mean number of shoots formed/propagule.
2. Mean shoot length (cm)/propagule.
3. Mean number of leaflets formed/propagule.
4. Mean number of roots formed/propagule.

RESULTS AND DISCUSSION

1. Surface sterilization

The obtained results in Table (1) indicated that using chlorox (commercial bleach) at 40% (v/v) achieved the highest percent survival (90%) compared to the other treatments which reflected on contamination percent (%), too.

Table (1). Effect of some sterilization treatments on the percentage of survival and contamination of Solidago canadensis explants in vitro after 35 days in culture.

Chlorox sodium Hypochlorite (NaOCl) Mercuric chloride (HgCl₂) which abbreviated as M.C.  

This finding may be accounted for the profound effect of this surface sterliant on elimination of external microorganism which could compete with the growth the given explants; subsequently, enhanced the growth of cultured propagules (Perkins, 1983). In this respect, using 1.00 mg/l of mercuric chloride, led to the highest percentage of survival of uncontaminated explants, i.e. 80% and 20% for contamination percent. The combination sterilants, chlorex and mercuric chloride at 40% and 1.00 mg/l, orderly; brought about was the highest percent of survival (100%) compared with all treatments. This finding could be attributed to that both disinfectant’s combination was toxic to microorganisms, but non-toxic for plant material.

Then, tissue cultures become possible with the use of convening and effective disinfectants such as alcohol and mercuric chloride (Pavan, 1999). Many investigators found that chlorox was the best treatments for explants disinfection (Mackay and Kitto, 1988; Mittal et al., 1989). Abd-Elrahman (2002) on Solidago altissima, confirmed the obtained results, whereas reported that the best result for surface sterilization was taken place due to using 40% clorox concentration (v/v) [NaOCl  at 2.0%], which produced the highest percentage of the free - contamined explants (80%) for"Toto" and (85%) var. "Tara" cultivar.  On the other hand, Beura et al. (2003) stated that in the case of axillary buds explants of Gladiolus cv.American Beauty, the treatment of 1% NaOCl for 10 min. was found to be the best; where it had 100% aseptic culture and survival of explants. Furthermore, Rozali et al. (2014) reported that the lowest contamination percentage of Calathea crotalifera was recorded when the cultured explanted were treated with combination of 30% (v/v) NaOCl, 70% (v/v) ethanol, and 0.3% (w/v) HgCl₂. In addition, Joshi et al. (2015) found that the best result of surface explants of Spilanthes acmella were surface sterilization using (1% and 4%) sodium hypochlorite and (0.01% and 0.1%) HgCl₂.

2. Initiation stage

Data presented in Table (2) and plate (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 ofSolidago canadensis 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 and callus formation in general, there was an inverse relationship between BA levels and the given traits, i.e. as BA level increased, the given trait decreased. However, the highest mean values were always recorded at the absence of either BA from the culture medium (nill level) or 0.25mg/l, but the lowest ones were noticed at its highest level (2.00 mg/l) except for the number of roots and callus formation which recorded at 0.00 mg/l. 

Table (2). Effect of different levels of BA and NAA (mg/l) and their combinations on the initiation stage of Solidago canadensis cultured in vitro for 35 days.

    L.S.D. (0.05) = Least significant difference test at 0.05 level of probability.

    *, **: Significant or highly significant.

Plate (1). Initiation stage of Solidago canadensis nodal explants cultured on MS medium supplemented with BA and NAA at 0.0 and 0.25 mg/l, respectively

Regarding the main effect of NAA tested levels on the above-mentioned traits, commonly; as NAA levels increased the given characters decreased. However, the highest mean values were always recorded at either the absence of NAA or at 0.50 mg/l for number of shoot, shoot length and number of leaflets formed per propagule. On the other side, the number of roots formation showed the highest mean values at 2.00 mg/l NAA added in the culture medium, but the lowest ones were noticed at its highest level (1.5 mg/l). The above-mentioned results indicated, generally, that decreasing the mean values of the studied characters was concomitant with increasing BA, could be attributed to accumulation supra-optimal level of cytokinins within tissues which exerts adverse effects on growth performance (Murashige, 1974; Tomas, 1987; George et al., 2008).

 Hence, Murashig and skoog (1962) medium without BA; resulted in the highest mean value of shoot length was taken place. As for NAA this finding could be attributed to the mode of action of enhanced auxin (NAA) either endogenously or exogenously within cultured tissues which is capable of controlling various distinctive processes such as cell growth and elongation (George and Sherrington, 1984 ; George et al., 2008). Furthermore, it is known that the role and mode of action of auxin for their abilities to enhance root formation was reported by many research workers (Chen et al., 1985; Liu et al., 1998; George et al., 2008; Waseem et al., 2011). Concerns to the interaction between both applied growth regulators at both 0.00 or 0.25 mg/l BA, and 0.00 and 0.50 mg/l NAA; expressed significant effects on the same tested traits.

3. Multiplication stage

Results in Table (3) and plate (2) describe the effect of various levels of both growth regulators and their combinations on the studied characters of Solidago canadensis. Respecting the main effect of BA, it showed that its presence in the culture medium at 0.50 mg/l; resulted in the highest mean number of shoots, shoot length and number of leaflets per propagule,  Meanwhile, its absence from the basal medium, led to the highest mean roots formed per propagule.

Table (3). Effect of different levels of BA and NAA (mg/l) and their combinations on the multiplication stage of Solidago canadensis cultured in vitro for 35 days

L.S.D. (0.05) = Least significant difference test at 0.05 level of probability.

*, **, N.S.: Significant, highly significant, or not significant, respective

Plate (2). Multiplication  stage of Solidago canadensis newly formed nodal segments during of initiation stage, upon culturing then on MS medium augmented with BA and NAA at 0.50 and 0. 50 mg/l, consecutively.

On the other end, the main effect of NAA declared that augmenting the basal medium with it at 0. 50 mg/l, brought about the highest number of shoots, shoot length and number of leaflets. However, its presence in the basal medium at 0.75 mg/l, give rise to the highest mean number of roots formed /propagule.  Meanwhile, the interaction between BA and NAA at 0.50 and 0.50 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 0.50 mg/l, was the optimal concentration for better performance of this particular hormone. Whereas, any above or lower deviation from this concentration of that growth regulator, the propagules showed poor performance. These results could be brought about to the mode of action of cytokinins on stimulation both cell division and promotion growth of axillary shoots in plant tissue culture as, also, found by Tomas (1987), Trigiano and Gray (2000) and George et al. (2008). These results are cope with Waseem et al. (2011) who reported that BAP as a cytokinin at the rate of 1.0 mg/l resulted in an increased number of shoots in Chrysanthemum morifolium formed per propagule. Abd El-rahman (2002) found that adding 0.5 mg/l BA produced the highest shootlet number for Solidago canadensis var. “Toto” and “Tara”.  Parihar et al. (2010) found that media supplemented with kinetin at 2.00 mg/l, was most effective for shoot induction, proliferation, and multiplication of Aegle marmelos.

In addition, Duhoky and Rasheed (2010) noticed that using kinetin at 2 mg/l, led to getting the highest length of new shoots of Gardenia Jasminoides. Gaber (2012) stated that the main effect of KIN showed its presence at the culture medium (2.0 mg/l), resulted in the highest mean shoot length. Meanwhile, its absence from the basal medium, led to the highest mean number of leaflets, nodes and roots formed per propagul. Regarding the role of NAA; the results, in general, showed that an intermediate concentration i.e.  0.50 mg/l of NAA produced the best results in almost tested traits. The fact that higher doses failed to manifest their superiority of their mode of action.

These findings could be attributed to an obnoxious effect at higher concentration. On the other extreme, the ineffectiveness of the lower dose indicate inadequate dose of hormone as a consequence indicating poor performance.  Likewise, this might be due to the mode of action of auxin (NAA) at the above-mentioned level within cultured tissues may enhance, control various distinctive processes such as cell growth and elongation (George and Sherrington, 1984). Wilkins (1989), additionally, stated that auxin induced number of responses which 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. However, the presence of auxins in the culture medium positively increased the mean shoot length of Trigonella foenum-graecum (Aasim et al., 2010). It is know 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 0.25 mg/l, brought about the highest shoot length.

 However, its presence in the basal medium at 0.125 mg/l, led to the highest mean number of leaflets, nodes and roots formed per propagule.  Also, El-Mahrouk 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).

 Furthermore, BA considers as antagonist for rhizogenesis and in favour of stimulates cell division, stimulates morphogenesis (shoot initiation/bud formation) in tissue culture and stimulates the growth of lateral buds-release of apical dominance. (Mauseth, 1991; Raven el al, 1992; Salisbury and Ross, 1992 and Davies, 1995). With respect to the combinations between both growths regulators (BA and NAA) led to significant effects on the studied traits.

4. Rhizogenesis

Results in Table (4) and plate (3) showed that the applied both auxin levels exerted significant effects on the studied characters of Solidago canadensis.

Table (4). Effect of different levels of IBA and NAA (mg/l) and their combinations on the rooting stage of Solidago canadensis cultured in vitro for 35 days.

L.S.D. (0.05) = Least significant difference test at 0.05 level of probability.

**: highly significant

Plate (3). Rhizogenesis of Solidago canadensismicroshoots of multiplication stage, upon culturing then on MS medium fortified with IBA and NAA at 1.5 and 2.00 mg/l, each in turn.

For IBA, the mean number of shoots at 0.50 mg/l, led to the highest mean values, and the main effect of NAA indicated that the absence of NAA recorded the highest mean value of the mean number of shoots.While, the interaction between IBA at 0.50 and NAA at nil (0.00) mg/l, resulted in 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 (nil) 0.00 mg/l. On the other side, the main effect of NAA at 0.00 mg/l recorded the highest mean value of shoot length. The interaction between IBA and NAA recorded no highly significant effects of the given trait at the absences of both IBA and NAA. Concerns of 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 had highly significant effect on the given trait at 0.00 mg/l. The interaction between both BA and NAA at 0.50 and 0.00 mg/l, respectively, resulted in the highest mean values .

Regarding the number of root formed per propagule, results showed that there is a proportional relationship between IBA and the given trait the highest mean value was recorded at 2.00 mg/l (5.58). On the other side, the main effect of NAA indicated that supplying MS-basal medium with NAA at 1.50 mg/l recorded the highest mean value of the mean number of roots (4.93). The interaction between IBA at 2.00 mg/l and NAA at 1.50 mg/l, gave the highest mean value of the given trait (6.89).

The obtained results showed that the used auxins (NAA and IBA), in general, produced the best results in almost all studied traits, except number of leaflets formed/propagule. These results could be explained on the bases that auxin induced number of responses which 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 (Wilkins, 1989).  However, the presence of auxins in the culture medium positively increased the mean shoot length of Trigonella foenum-graecum (Aasim et al., 2010). On the other end, the above-mentioned of auxin results indicated a marked superiority on root formation, whereas the highest concentration i.e. IBA at 2.0 mg/l, let to the highest number of roots formed /propagule. Moreover, IBA at the above-mentioned level confirmed its superiority over NAA. The reason for these differences in root inducing ability may be due to the slow and continuous release of IAA than IBA (Krieken et al., 1993 and Liu et al., 1998) and release of IBA through hydrolysis of conjugates (Epstein and Muller, 1993). These IBA conjugates were reported to be superior to free IBA in serving as an auxin source during later stages of rooting (Staswick et al., 2005).  However, similar results regarding chrysanthemum were obtained by Karim et al. (2002) and Long et al. (2006). Also, Hoque et al. (1995) on C. morifolium, reported thatmaximum number of roots was obtained on MS media supplemented with IBA compare to NAA. Whereas, Long et al. (2006) stated that the highest number of roots were obtained when chrysanthemum microshoot cuttings were treated with IBA. Waseem (2008) reported that IBA showed its superiority over all other tested auxins (viz. NAA and IAA), when used alone. Litz and Jaiswal (1990) mentioned that IBA is considered as the most effective auxin for root induction. Our results were further confirmed by the previous findings of Komalavalli and Rao (2000); Sarker and Shaheen (2001); Munshi et al. (2004); Awal et al. (2005); Rajani and Patil (2009) and Waseem et al. (2011) who suggested that IBA is the best auxin for root induction and development.  Further confirmations, in this respect, were reported by Benelli et al., (2001) and Tanimoto (2005) who proved that IBA is the most effective auxin in olive rhizogenesis as compared to NAA. The inferior effect of NAA on the root number may be due to the reason that NAA is more persistent than IBA, remains present in the tissue and may block further development of root meristemoids (De Klerk et al., 1997 and Nanda et al., 2004).

Regarding to the interaction between both applied growth regulators between IBA at 0.50 mg/l and NAA at (nil) 0.00 mg/l, led to significant effects on the almost of vegetative growth. On the other hand, the interaction between IBA at 2.0 mg/l and NAA at 1.5 mg/l, led to good performance to enhance rhizogenesis.

5. Ex vitro and in vivo acclimatization of Solidago canadensis

Data presented in Table (5) 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 single node explants ofSolidago canadensis grown ex vitro for four weeks and as shown in Figure (4).

Table (5). The effect of different potting mixtures of perlite and peatmoss (v/v) and their combination on the acclimatization of neoformed plantlets of Solidago canadensis after four weeks ex vitro.

L.S.D. (0.05) = Least significant difference test at 0.05 level of probability.

**:  highly significant.  

 Plate (4). Acclimization of neoformed Solidago canadensisunder combination   of perlite and peatmoss at (1: 3 v/v)

Concerning the average of survival percentage/ plant, perlite had a highly significant effect on this trait. The highest mean value was recorded at level 1v/v (83%). Also, peatmoss had highly significant effect on the graven traits was recorded at level 3v/v (86%). Meanwhile, the interaction between peatmoss and perlite exerted highly significant effect. However, the combination of perlite and peatmoss at either 1:1or 1: 2 and 1:3, respectively, resulted in the highest mean value (100%). Respecting the average number of neoformed shoots / plant, perlite and peatmoss and their interactions, exerted significant effects on the given trait. 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 3 v/v. However, the interaction between both added levels of 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. Abd –Elrahman (2002) reported that the plantlets (5-6 cm) resulted from the previous in vitro culture of explants were transferred for four weeks into 0.2liter-capacity pots containing peatmoss with 0.2% Topsin- M70 fungicidal solution in therane-house; whereas, the culture pots  were covered with transparent polyethylene bags. One week later, the polyethylene bags were repeatedly perforated with one hole for, four times at week intervals. After two weeks, the polyethylene bags were totally removed before transplanting out of door. At the end of acclimatization period (after four weeks), acclimatized plantlets were grown successfully and produced high survival percentage ranging from 97-99%. 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. Also, Paul et al., (2013) stated that in vitro-raised plantlets were hardened in polycups containing a mixture of sterile garden soil: sand (3:1), covered with polypropylene bags and irrigated with 10 X diluted MS liquid medium. The plants were kept in a culture room for 15 days then 63% of plants were successfully established in polycups. After 15 days the polycups hardened plants were transferred to pots and kept in greenhouse. However, 74% percent of plants were well established under greenhouse condition. After one month, the plants were transferred to the field. About 67% of plants were established in the field.In conclusion, this study established such techniques to propagate solidago canadensis in vitro and acclimatization ex vitro successfully.