In Vitro Propagation and Ex Vitro Acclimatization of Magnolia (Magnolia grandiflora, Linn) Trees

INTRODUCTION

Magnolia grandiflora, Linn which also called evergreen  magnolia, bull boy, or large flower magnolia,  is evergreen  tree.  It is pyramidal tree with creamy flowers belongs to family Magnoliaceae (Chaidaroon et al., 2004; Said, 2007). There are at least 100 species which share genus magnolia. It was first grown as an ornamental evergreen tree in the world in southern U.S. A.  It is growen in Egypt in botanical and private gardens. It was quickly popularized for its glossy evergreen foliage, large beautiful flowers and elegant form and extensively planted as an ornamental plant (Said, 2007).  Magnolia  grandiflora bears large, very fragrant, bowl- shaped, white flowers.They show intermittently from mid-summer to early autumn  (Bailey  and Bailey ,1960) . It is, also, used a specimen plant, shade trees, screen or wind break, it can be grown as esplalier. It is valuable for garden or park planting in Japan. Its wood is used for furniture and various industrial arts, and the bark contains valuable medicinal compounds (Nakmura et al., 1995).

In Alexandria (Egypt), Magnolia  grandiflora  trees are grown in private  and botanical gardens  with a  alimented. It is, known that Magnolia grandiflora is propagating by such vegetatively propagated methods as Averages seeds, but its lower germinability rate (ca.35%), or by cutting but it, also, difficult because of its poor rooting ability (Nakmura et al., 1995) or by air layering, but it is labor extensive and wasting time- method, as well as very expensive (Mccracken  et al.,1996)  Further, rooting of this species is too -hard to achieve. Also, the demands of Magnolia grangiflora for rooting facilities are very much expensive and may be unavailable to root shoot cuts (Said, 2007).   Therefore, propagation of Magnolia using plant tissue culture   techniques may offer certain advantages over traditional method of propagation. It is one of the most promising and advanced applications of plant cell and tissue culture technology to propagate this species in vitro. This technique could be very useful to provide hundreds or thousands of this species withen a limited time.

Therefore, the present study was aimed to establish an efficient and reliable protocol for in vitro propagation and with focusing on rhizogenesis of this hard- to –root species.

MATERIALS AND METHODS

Plant material and explants sterilization

The plant material was collected from tree grown in garden of Ornamental and Landscape, of the Research Department of EL-Montazh, Alexandria, Egypt. The tree was sprayed with the fungicide and insecticide 2-3 week prior to start initiation and over head watering was strictly avoided. Freshly grown shoot tips, with two to three nodes, were selected as explants’ source. The collected material was brought to the Plant Tissue Culture Laboratory of the Plant Production Department of the Faculty of Agriculture, Saba Basha, Alexandria University during 2013-2015 seasons and washed, thoroughly, with running tap water for 30 minutes to remove the dust or sand particles. The explants were cut to nodal segments (single node) as an explants’ source (Bhattacharya et al., 1990).The excised explants were dipped in 70% ethanol, for 1 min. after treatment with ethanol the explants were rinsed with double distilled water twice, so as to lower the toxic effect of ethanol. Nodal segments of magnolia were surface sterilized with sodium hypochlorite (NaOCl) solution (commercial bleach as ‘clorox’) at 30% for 20 minutes followed by mercuric chloride  at 1.5mg/l for 5min with few drops of Tween-20, also, were added as a surfactant to the sterilized water . Finally, they were washed three times with sterile distilled water and became ready for culture.

Microprogation stages

Intiation stage

The explants were cultured on solidified woody plant medium coined as WPM (Lloyd and McCown, 1980) which contained different concentrations of kintine (KIN) at four concentrations:0.0(nil), 0.5,1.0 and 2.0 mg/l, in combinations with the auxin Indole butric acid (IBA) at four concentrations: 0.0(nil) ,1.0, 2.0 and 3.0 mg/l. Three explants were cultured in each jar which containing 30ml of medium and were placed, vertically. Each treatment was replicated three times and each has 3 explants (i.e.9 explants /treatment).The jars were capped with aluminum foil closures. The cultured jars were incubated in growth chamber at 25 C temperature under 16 hr daily light and 8hr darkness illumination by a florescent light intensity of 2880 Lux (40 mol ) at 97%R.H.

Multiplication stage

The neoformed propagule of the initiation stage was sectioned into single leaflet node. The excised nodal cutting explants of the different positions were cultured, randomly, onto the multiplication medium (WPM) supplemented with KIN at four concentrations: 0.00(nil), 1.00, 3.00 and 5.00 mg/l, in combinations with IBA at four concentrations:0.00(nil) ,0.50, 1.00 and 2.00 mg/l.

Rooting (rhizogenesis) stage

The obtained shoots of magnolia from the multiplication stages were, individually, separated and cultured on a rooting medium for rhizogenesis to achieve this stage .This medium was augmented with two types of auxins which were used as Indole Butyric Acid (IBA) at four concentrations: 0.00(nil), 0.50, 1.00 and 2.00 mg/l, in combinations with NAA at four concentrations: 0.00, 1.00, 2.00 and 3.00 mg/l. Generally, the data were recorded per propagule at initiation, multiplication and rooting stages after 35 days in culture. The tested characters were as follows:

-Average shoots length (cm)/propagule.

-Average number of shoots formed/ propagule.

-Average number of leaflets formed/ propagule

-Average number of roots formed/ propagule.

Comparison between the effect of BA and KIN on explants ofmagnolia

The comparison between the effect BA(Benzyl adenine) and KIN(Kintine) on growth of Magnolia  during multiplication  stage was investigated  in a 5x3 factorial experiment in which the treatment at 3.00mg/l , each in trun.

Effects of activated charcoal on shoots plantlets

Activated charcoal has been used previously to adsorb ethylene and other growth inhibitory substances produced in shoot cultures derived from the medium or from the plant tissues or both. Four main treatments were tested :no addition of activated charcoal(AC) to the medium ,and three concentrations of 0.25,0.50 and1.00g/l, (AC).The media pH was adjusted to 5.7, then  gerlrite at 7g/l was added before autoclaving .Each treatments was replicated 3 times and each replication has 3 explants. The followed characters were recorded per propagule after 35 days in culture:

1-Average number of shoots formed per propagule.

2-Average shoots length (cm) per propagule.

3- Average number of roots formed per propagule.

4-Average number of leaflets formed per propagule.

Acclimatization stage

The new formed plantlets (rooted shoots) were then transferred to the greenhouse for hardening. The potting mix used in this study comprised of sand and compost (1:3) .The transferred plants were monitored weekly for at least 6 weeks.

Statistical analysis

A completely randomized design was used for all the experiments (Gomez and Gomez, 1984).Recorded data were analyzed, statistically, using analysis of variance technique (ANOVA) and averages were compared by the least significant difference (L.S.D.) (Steel et al., 1997) and significance was determined at p 0.05.

Results and Discussion

Achievement of optimal and reliable system for micropropagation of Magnolia grandiflora was urgent and in focus. Therefore, a set of experiments was conducted, and the obtained results were presented and discussed in the following section as follows:

Micropropagation

Initiation stage

Data outlined in Table (1) exhibit that  both applied growth regulator, levels and their combinations exerted, highly, significant effects on the initiation stage characters of Magnolia. Single node explants were grown in vitro for 35 days as shown in Figure (1).

Concerning the main effect of studied cytokinin (KIN), in terms of the Average shoot length/propagule, supplementing the culture medium with at 0.5mg/l; resulted in the highest Average value (2.11cm), compare with the other treatments. On the other hand, augmenting the culture medium with IBA at 2.00 mg/l was concomitant with the highest Average value of the given trait (2.60cm).

In addition, the interaction between KIN at either nil level (0.00) or 0.5 mg/l of KIN with IBA at 2.00mg/l, brought about the highest Average values of the studied trait (i.e. 2.96 and 3.00 cm) each in turn.

Respecting the Average number of shoots formed/propagule, fortifying the culture medium with KIN, led to remarkable notes; where, as KIN levels increased the Average value of the given trait increased. Whereas, adding the highest level, 2.00mg/l KIN; gave rise to the highest Average value of the studied character (1.78) and vice versa. As for the main effect of IBA ,it is obvious that augmenting the culture medium with it at 1.00mg/l, contributed in achieving the highest Average value of this trait (1.68) compare to other treatments. Mean while, the interaction between KIN and IBA at 2.00 mg/l, and either of added levels of IBA led to the highest Average values, with siginificant difference. With reference to the Average number of leaflets formed/propagule, the main effect of KIN was obvious through adding it at 1.00mg/l, which recorded the highest Average value of the given trait (3.77) compare to the other treatments .On the other hand,  the main effect of IBA declared that adding 2.00mg/l of IBA to the culture medium ; achieved the highest Average  value (3.72) compare to the other tested levels. Also, the interaction between KIN  and IBA at various combinations, especially at 1.00 and 2.00 mg/l, each in turn, achieved the highest Average value, but without significant differences . Refer to the Average number of roots formed /propagule, the KIN levels were in reverse relationship with this studied trait.  Whereas, as KIN levels increased the given trait decreased. Hence, the absence of KIN in culture medium led to the highest Average number of roots formed/propagule (1.36) compare to the other treatments. On the other tank, the main effect of IBA was obvious especially at its presence in culture medium at 2.00 mg/l, recorded the highest

                        IBA             KIN levels (mg/l)        Average       Significance

Characters    levels     0.00   0.50   1.00   2.00         IBA        KIN   IBA  KINXIBA

(a)Average shoot length (cm)/propagule:

                       0.00        0.91   0.96   0.90   0.83         0.90     **         **          **

                       1.00        1.93   1.96   1.83   1.73         1.86

                       2.00        2.96   3.00   2.30   2.13         2.60

Average(KIN)                1.99   2.11   1.82   1.70

 (b) Average number of shoots formed /propagule:

                       0.00        0.67   1.20   1.26   1.56         1.17      **                **            **

                       1.00        1.26   1.76   1.83   1.86         1.68

                       2.00        1.26   1.36   1.66   1.86         1.54

Average (KIN)               1.05   1.40   1.54   1.78

 (c) Average number of leaflets formed /propagule:

                       0.00        1.70   3.81   3.83   3.93         3.32      **                   **             **

                       1.00        3.23   3.70   3.87   3.71         3.62

                       2.00        3.60   3.75   3.96   3.57         3.72

Average (KIN)               2.98   3.66   3.77   3.63

 (d) Average number of roots formed /propagule:

                       0.00         0.01   0.33    0.33   0.00         0.16      **       **  **        

                       1.00         1.81   0.73   0.37   0.36          0.82

                       2.00         2.32   1.67   0.66   0.56          1.30

Average (KIN)                1.36   0.93   0.59   0.48

L.S.D._(0.05)= Least Significant Difference test at 0.05 level of probability.*, ** Significant of highly significant.

Fig.(1):Intiation of magnolia nodal explants cultured for 35 days on WP   medium supplemented with KIN (2.00mg/l) and IBA at 1.00 mg/l.

average value (1.30). While, the interaction between KIN and IBA at nil level (0.00) and 2.00mg/l, consecutively, achieved the highest Average value (2.32).  The obtained results in this respect are matching with the mode of actions  of both applied growth regulators; whereas, auxin  exerts significant roles in plant tissue culture and usually form an integral part of nutrient media. Auxin promotes either individually or in combination with cytokinins, the growth of calli, cell suspensions and organs and also regulatate the direction of morphogenesis. At the cellular level, auxins control basis processes such as cell division and cell elongation (George et al., 2008).Also, they play critical event in promoting rhizogenesis (Kim et al.,2003).

Cytokinins, together with auxin, take part in the regulation of the cell cycle in plant cells (i.e. stimulation of cell division, break apical dominance, enhance axillary shoot proliferation, and adventitious, inhibition root formation). Also, the interaction between auxin and cytokinin or their ratio other reperesents an important signal in the formation of cell phenotype and in the onest and maintenance of the process of cell division (Stickens et al., 1996).The ability of auxins (together with cytokinins) to mange key events in plant morphogenesis was documented (Skoog and Miller,1957) who discovered the regulation of organogenesis in vitro by Averages of the auxin: cytokinin ratio in culture media. It has been further supported by such other researches on the relationship between auxin and cytokinin levels and the morphogenetic response of various plants (Li et al., 1994; Centeno et al., 1996; Leyser et al., 1996).

The higher concentrations of the auxins as NAA is usually ineffective against shoot proliferation (Vijaya et al., 1991, Waseem et al., 2011). Results of this study clearly demonstrated that WP medium was a better choice and improved growth of cultured explants (nodal segments) as reported by Biedermann (1987). It is noticeable that using of IBA at 2.0 mg/l here seamed to promote elongation of shoots that was considered an added help for the survival and growth of shoots. Similar results, during the establishment stage, were reported, elsewhere, on Magnolia when IBA was employed (Franc and Krejci, 1998). EL-Shamy (2004 and EL-Shamy et al. 2010) reported that subcultures on MS medium supplemented with 2.0 mg/l NAA led to increase the shoot length and number of leaves. With regard to IBA concentration, the addition of IBA at 1 mg/l to B5 medium resulted in the highest number of shoots. Raising the level of IBA to 2.0 mg/l, significantly, decreased the degree of browning induced callus formaition and gave the longest axillary shoots (Sakr et al.,1999). The induction rate of Magnolia officinalis was 100% on Gamborg medium (B5) containing 4.0 mg/l 2, 4-D and 1.0 mg NAA/ litre. The highest proliferation rate and the lowest percentage of callus browning were recorded from B5 medium containing 1.2-2.0 mg BA and 1.0 mg NAA/l (Tong et al., 2002). On the other side, it was found that the lower the salt concentration, the more shoot elongation of Magnolia was hampered and the better the root formation. Higher KIN levels (2.5 mg/l) in combination with high salt concentration (1/1 and1/2) allow fairly uniform elongation shoot while rooting was poor. On low salt media, rooting was prominent but the leaves were yellowing and the elongation was nil (Maene and Debergh, 1985). The best results were obtained with mill medium + 1.0 mg IBA/ l (average number of

shoots 4.6, average length of shoots 5.0 mm. Krejci and Franc,1997).  Qi et al. (2010) reported the terminal buds of Magnolia officinalis cultured on MS medium plus 6-BA, NAA and 2,4-D under different light conditions. The results showed that the optimum medium for callus indication of Magnolia officinalis terminal buds is MS+2.0 mg/l 2,4-D+ 0.5 mg/l BA+1.0 mg/l NAA +30 g/l sucrose+8 g/l agar at pH 5.8. Also,  Li and Dong (2007) found that the terminal bud of the lower branches of M.Simicum should be used as explants in a medium consisting of ½ MS medium with 0.1- 1.5 mg/l IBA and 0.1- 1.0 mg/l IBA. Angsumalee et al. (2005) reported that all the explants formed viable shoots when MS medium supplemented with 0.1- 10 mg BA /l. Shooting was highest (2.72±0.37) on MS medium supplemented with 1 mg/l for 4 weeks. For successful in vitro rooting, shoots of Magnolia grandiflora were treated with 2.00 mg/l IBA in the culture medium. Whereas, lesser or higher IBA concentrations (i.e.1.00 mg/l or 3.0 mg/l) failed to form roots on shoots. Similarly in other trials, 2.0 mg/l IBA promoted in vitro rooting on Magnolia shoots (Maene and Debergh, 1985; Kamenicka et al., 1996, Sakr et al., 1999, El-Shamy et al., 2004 and 2010). In some other cases, IAA was also used for in vitro rooting of Magnolia shoots (Kamenicka and Takats, 1997).

Multiplication stage

Results of Table (2) and Figure (2) display  the effect of both applied growth regulator’ levels and their combinations practiced highly, significant effects on the multiplication stage’s characters of Magnolia grandiflora where single nodal explants were cultured and grown in vitro for 35 days . Respecting the shoot length formed per propagule, the main effect of KIN  declared that increasing levels’ concentrations within the range of1.00- 3.00mg/l with no significant difference, led to increase the shoot length at but increasing its level up to at 5.00mg/l, coused such significant decrease. On the other hand, the main effect of IBA, declared that there was a proportional relationship between it and the given trait. Whereas, the IBA at 1.0 mg/l and/or at 2.00mg/l, resulted in the highest Average values (2.44 and/or 2.48) without significant difference. Regarding the interaction between both applied growth regulators, the presence of two hormones KIN and IBA at 1.00 mg/l each, resulted in the highest number of shoots per propagule (2.85). Respecting the Average number of shoots formed/propagule, the main effect of KIN showed that its presence in the culture medium at 5.00mg/l achieved the highest Average value (2.88).On the other extreme, the presence of IBA in culture medium at either 1.00 or 2.00mg/l, brought about the highest Average values, i.e.2.44 or 2.48, without significant difference. The interaction between KIN and IBA at 5.00 and 1.00 mg/l, respectively recorded the highest Average value (3.40). In terms of Average number of leaflets formed/propagule, adding KIN to the culture medium at 5.00mg/l contributed to record the highest Average value of the studied trait (6.58). Respecting the main effect of IBA, augmenting the culture medium with either 1.00 or 2.00mg/l, resulted in similar finding; where achieved either 5.44 or 5.35 respectively, without siginificant difference. Meanwhile, the interaction between KIN and IBA at 5.00 and 1.00mg/l, led to the highest Average value of the given trait (7.33). With respect of the Average number of roots formed/propagule, it is obvious that the absence of KIN from the culture medium (0.00mg/l), gave rise to the highest Average number of roots formed/propagule(1.18), then as its level increased in the culture medium, the given trait was in inverse relationship. On the other hand, augmenting the culture medium with IBA was in direct propontional relationship with the given trait, especially at 2.00mg/l which achieved the highest Average value (1.33). Meanwhile, the interaction between 0.00mg/l and 2.00mg/l of both KIN and IBA, each in turn, brought about the highest Average value (2.00 roots per propagule).

                       IBA             KIN levels (mg/l)         Average             significance

Character    levels     0.00   1.00    3.00    5.00      IBA         KIN    IBA KIN X  IBA  

(a)Average shoot length(cm)/propagule:                             

                       0.00      1.06   1.26   1.23   1.20     1.19             **      **        **

                       0.50      1.30   1.33   1.41   1.33     1.34

                       1.00      1.63   2.85   2.63   2.66      2.44

Average (KIN)             1.63   1.99   1.99   1.83

L.S.D.(0.05)                                                                           0.10    0.10    0.21

 (b) Average number of shoots formed /propagule:

                       0.00        0.85   1.23   2.63   2.73      1.86         **      **         **

                       0.50        1.00   1.57   2.06   2.85      1.87

                       1.00        1.32   1.91   2.33   3.40       2.24

 Average (KIN)               1.18   1.67   2.29   2.88

L.S.D.(0.05)                                                                            0.14    0.14      0.29     

(c) Average number of leaflets formed /propagule:

                      0.00       2.36   4.78    5.80   6.25        4.80       **         **          **

     0.50       2.93   4.16    5.25   6.66        4.75

     1.00       3.50   4.58    6.36   7.33        5.44

     2.00       3.83   5.50    6.00   6.08        5.35

(d) Average number of roots formed /propagule:          

                      0.00      0.00   0.33    0.33   0.00         0.16      **         **          **  

                      0.50      1.00   0.56    0.44   0.00         0.50

                      1.00      1.72   0.70    0.67   0.55         0.91

                      2.00      2.00   1.33    1.00   1.00         1.33

L.S.D.(0.05)                                                                            0.06      0.06    0.11     

L.S.D._(0.05)= Least Significant Difference test at 0.05 level of probability.*, ** Significant of highly significant.

Figure (2): Multiplication of magnolia from newly nodal segments of initiation stage, upon cuturing for 35 days on WPM augmented with KIN and IBA at 5.00 and 1.00mg/l, consecutively

On the other side, Magnolia grandiflora, also, responded positively to form callus by application of KIN during the multiplication stage (Klimazewska, 1981). In the multiplication stage, adding 5.0 mg/l KIN culture medium the c formed the highest number of shoots as reported by EL-Shamy et al.(2010). This finding could be achieved due to the mode of action of auxin (IBA) within cultured tissues which many enhance, control various distinctive processes such as cell growth and elongation (George and Sherrington, 1984) and Wilkins (1989). Additionally, it has been stated that auxin induced number of response which involved cell division,cell enlargement, protein and nucleic acids synthesis which are concenation of auxin – induced  growth and changes in wall plasticity of plant cell and increase the apical dominance as there are assential and rapid processes involved in growth and elongation. Howaever, the presence of auxin in the culture medium, positively, increased the Average shoot length of Magnolia grandiflora (Saker et al., 1999; Zaman et al., 2001; EL-Shamy et al., 2010).  In this respect, also, Lemos and Black (1996) showed in Annona muricata that the addition of NAA promoted bud elongation.  The more important multiplication stage, the use of KIN favoured not only proliferation of shoots, but also promoted plant height of magnolia shoots. Whereas, KIN at 5 mg/l led to the highest number of shoots and at 1.00 or 3.00 mg/l led to the tallest plant heights, number of leaves was restricted to leaf surrounding the formed bud only at 1.00 or 3.00 mg/l of KIN. When KIN was used in a lesser concentration (0.00mg/l or 1.00mg/l) number of leaves were dramatically decreased. However, MS supplemented with NAA or IAA was used successfully with magnolia for shoot proliferation purposes (Krejci and Franc,1997). However, in other occasions Magnolia, also, responded positively to form callus by application of KIN during the multiplication stage (Klimaszowska, 1981). This later auther reported that the obtained result was in harmony with Magnolia grandiflora results obtained here, which on the whole, seems to favour KIN for the multiplication stage of Magnolia. As an explanation for this phenomenon, its more likely that high levels of KIN- utilized in this study (i.e. 5.0 mg/l) and elsewhere too, may have caused the removal of apical dominance thus enhanced shoot proliferation (Klimaszewska,1981). In this respect, in Magnolia grandiflora, it was, also, observed that shoot multiplication was developed using different concentration of cytokinin with auxin achieved the best results as reported by some investigators  (Nakmura et al., 1995; Luo and Sung,1996;  Tong et al., 2002; Zaikang et al., 2002;Rosas and Rodriques, 2006; Lina et al.,2006 and Parris et al., 2010).

Rooting (rhizogenesis) stage

Since the growth and development are correlated processes the recorded characters, here, should be presented as a whole.

Results of Table (3) and Figure (3) manifested that various levels of both applied growth regulators and their interactions had, highly significant effects on the rooting stage’s traits of Magnolia grandiflora. Respecting the Average shoot length per propagule, results of IBA demonstrated that the presence of IBA in culture medium results in the highest shoot length. In total, the main effect of NAA showed similar performance that has been noticed as the above- mentioned characters. The interaction between IBA at 1.00mg/l and NAA at 2.00 mg/l gave the highest Average value of shoot length (2.31). It could be concluded from the above-mentioned results that the presence of IBA in the culture medium led to better performance of shoot length . Higher concentration of IBA had shown best results. This reason could be due to the fact that IBA as usually take an active role in the shoot proliferation and its effect is visible in callus or root formation. On the contrary, NAA at intermediate concentration (viz 2.00 mg/l), caused the longest shoot per propagule. The finding could be attributed to the mode of action of auxin (NAA) within cultured tissues is capable of controlling various distinctive processes such as cell growth and elongation (George and Sherrington, 1984; George et al., 2008). Concerns for the Average number of shoots per propagule, the main effect of IBA .

Fig.(3):Rhizogenesis of magnolia microshoots of multiplication stage, upon culturing then for 35 days on WP medium fortifited with NAA and IBA at 1.00  mg/l, each in turn

Table (3). Effect of different levels of NAA and IBA(mg/l) and their combinations on the rooting stage of Magnolia grandiflora nodal cuttings cultured in vitro for 35 days.

Characters    levels       0.00   1.00   2.00   3.00       IBA     NAA   IBA  NAAXIBA

(a)Average shoots length (cm) /propagule:

                       0.00        1.00   1.41   1.60   1.76         1.44     * *     **         **

                       0.50        1.65   1.80   1.90   1.98         1.83

                       1.00        1.95   2.03   2.31   1.91         2.05

Average (NAA)              1.64   1.81   1.95   1.81

 (b)Average number of shoots formed /propagule:

                       0.00        0.66   1.64   1.33   1.03         1.16     **      **          **

                       0.50        1.33   1.46   1.39   1.30         1.37

                       1.00        1.63   1.86   1.34   1.23         1.51

Average (NAA)              1.23   1.59   1.29   1.14

(c) Average number of leaflets formed /propagule:

                       0.00        2.32   3.18   3.23   3.16         2.97     **       **         **

                       0.50        2.62   3.30   3.26   3.28         3.11

                       1.00        2.73   3.34   3.38   3.18         3.16

Average (NAA)              2.62   3.21   3.21   3.09

(d) Average number of roots formed /propagule:

                       0.00        0.20   1.00   1.60   2.33         1.28     **         **           **

                       0.05        1.26   1.33   1.80   2.66         1.76

                       1.00        1.85   2.83   2.60   2.46         2.43

Average (NAA)              1.39   1.92   2.05   2.36

L.S.D _(0.05)= Least Significant Difference test at 0.05 level of probability*, ** Significant of highly significant.

and NAA, divulged that the presence of both growth regulators-into WP medium had significant effects of the given trait. The interaction between NAA and IBA exerted, highly, significant effects and at the highest levels of both the growth regulators brought about the lowest Average values. Concerning the effect of IBA on the rooting mass perpagule, it is obvious that, the fortifying WP-medium with IBA at 1.00 mg/l, results in the highest Average value. On the other way, the presence of NAA into culture medium at 3.00 mg/l, brought about the highest Average value. Likewise, the combinations between both IBA and NAA at 1.00 mg/leach, gave the highest Average values. These results showed that the medium fortified with 1.00 mg/l IBA, and NAA at 3.00 mg brought about the highest rooting mass per propagule. These results cope with those of microcuttings of Magnolia soulangiana were treated in vitro with 0.1 , 1.0 , 2.0, 3.0  or 4.0 mg IBA . Root number was greatest with 4.0 mg IBA and root length with 1.0 mg IBA (Kamenicka, 1996). In the later, trial, the optimal rooting medium for Magnolia was half stermsth s-medium with 4 mg IAA/l (Kamenicka and Takats, 1997). Similar results were reported by Chaidaroom et al.(2004). Concenring, the main effect of IBA tested levels on the Average number of leaves per propagule, the presence of IBA at 1.00 mg/l, led to the highest Average values of above - mentioned traits. On the other hand, NAA main effect, augmenting WP- basal medium with 1.00 or 2.00 mg/l of it, brought about the highest Average value of the above - mentioned traits. However, the interaction between both added levels of IBA and NAA at 1.00 mg/l, resulted in the highest Average value.

Comparison between the effect of BA and KIN on explants ofMagnolia   grandiflora

 As for data presented in Table (4) and Figure(4) declared that the effectiveness of KIN surpassed significantly its counterpart of BA (at 3.00mg/l =13.33  each).  Regarding shoot length and number of shoots/propagule (viz., 2.67 and 2.31viz. 1.90 and 1.20, each in turn). Meanwhile, there were insignificant differences respecting number of leaftlets and number of shoots/propagule. These results could be attributed to the mode of action of KIN which is more effective than BA and /or variations in their metabolism or to active forms or to differences in primary mechanism of action as reported earlier.  Alternatively, responses of explants to both cytokinins are different due to various aspects. Also, this variation may be due to the degree of cell sensitivity towards both tested cytokinins, which depends on the endogenous levels of growth regulators. Likewise, in other occasions, BA was reported to be not suitable for Magnolia elongation in the multiplication stage (Maene and Debergh, 1985; Biedermann,1987; Kamenicka el al.1996; Luo and Sung,1996; Kamenicka and Takats,1997). Magnolia, also, responded positively to form callus by application of KIN during the multiplication stage (Klimaszewska, 1981). This later reported result is in harmony with Magnolia grandiflora results obtained here, which on the whole, seems to favour KIN for the multiplication stage of Magnolia .As an explanation for this phenomenon, it is more likely that high levels of KIN utilized in this study (3.00mg/l) and elsewhere, too, may have caused the removal of apical dominace thus enhancing lateral shoot proliferation (Klimazewska, 1981).Also, EL-Shamy(2004)reported that Magnolia grandiflora at the multiplication stage, the best medium was WP medium plus the growth regulators KIN (at 5.00 or 6.00mg/l) which increased plant height, number of leaves/shoot and number of shoot .Notably, KIN was better than BA for the multiplication stage of Magnolia grandiflora.

Table(4). Effect of both growth regulators (BA and KIN) at 3.00mg/l each on Magnolia grandiflora explants grown in vitro for 35 days/propagule during multiplication stage

Figure (4): Effect of BA (left) and KIN (right) at 3.00mglL, each on growth performance of magnolia grown in vitro for 35 days

Effects of activated charcoal on Magnolia

Data presented in Table (5) and Figure (5) showed that adding activated charcoal to WP medium resulted in high significant effects on the given traits . However there were  direct proportional relationships between concentrations of AC and the all given traits, especially at 1.00 g/l AC. The role of activited charcoal in tissue culture is interpreted by Fridborg et al.(1978) as being an adsorbent for inhibitory materials that may be persented in the medium or that might originate from the explants themeseleves .Also, charcoal is characterized by the adsorption of toxic brown or black pigments (phenol-like compounds and melanin) and absorption of all other organic compounds, auxin, cytokinin, ethylene, vitamins Fe and Zn chelates (Pierik, 1987).Fridberg et al.(1978) found that compounds excreted from growing cells of Daucus  and Allium could be adsorbed by activated charcoal to allow embryogenesis and root formation to occure. On Ruscus  hypoglossum plants Abou-Dahab et al. (2005) decided that the highest number of roots was recorded with 5g /l sucrose and with activated charcoal. Parris et al.( 2010)  found that supplemented McCown woody plant medium (WPM) with charcoal produced elongated  plantltes which more sutitable for increase rooting and ex vitro establishment  on Magnolia “Ann” .Also, Thomas (2008) reported that  increased shoot elongation Acacia mearnii and Anacardium. Gad (2011)reported that shootlets of Populus alba were  elongated on medium supplemented with 3mg/l  +3g/l AC+0.5 mg/l BAP followed by highest rooting on medium with 3mg/l +3g/l AC+0.1mg/l NAA +0.5mg// IBA. Then, Charcoal can increase the capability of culture shoots to form more roots.

                      Activated charcoal concentrations      significant

(a)Average shoot          1.39         1.93     2.60      3.31           **             0.20

Length (cm)/propagule                                                                                                       

(b)Average number of   1.50         1.62     2.00      2.96           **            0.16

shoots formed propagule                                                                                                   

(c)Average number of    3.03         3.00     5.30     5.83            **           1.20

Leaflets formed/propagule                                                                                                 

(d)Average number of    0.52         1.13     1.86     2.06            **           0.45

L.S.D.(0.05)=Least significant different test at 0.05 level of probability*,**: significant or highly significant

         (a)                        (b)                      (c)                       (d)

 Figure (5): Effect of activated (AC) charcoal concentrations on the growth of magnolia, (a)control ; (b)0. 25 g/l ; (c)0.50g/l ,and (d)1.00 g/l

The fourth stage (acclimatization)

Acclimatization of in vitro grown plants is an important step in micropropagation (Smart, 2008; Rout et al., 2006).The In vitro grown plantlets with at least two to three roots were transferred to the greenhouse for the acclimatization ex vitro. The potting mix (sand and compost, 1:3), routinely used in the nursery of our institute, was found suitable for the hardening of the plants. The survival rate of the In vitro grown plants was 40% as shown in Fig. (6).

Figure (6): Acclimatization of Magnolia plantlets ex vitro in a mixture of compost and sand (3:1)

Further prospects (recommendations)

The present study declared the necessity for further studies to improve

(1)proliferation or multiplication of neoformed shoots; (2) achieving both proliferation or multiplication and rooting at the same time via a dual stage culture medium for saving the time and efforts to achieve each stage indivivally ; (3) in addition find out the way through to overcome decline multiplication rates with heading the subculture via resting the multiplied shoots over subculture (culture on media without growth regulators or half concentration……ect., and(4) improving the survival of magnolia plantlets ex vitro and in vivo, via choosing the appropriate mixtures.

Conclusion

It could be concluded that there is a possibility to propagate magnolia trees by micropropagation.  The protocol here in descried is very much efficient for the in vitro intiation (elongation of regenerated shoots), multiplication shoot prolifiration, rooting of nodels egements of this species  (hard -to –root plants).

In the light of our results:

1- It can be suggested that enhanced shoots and buds formation can be achieved by using the WP media fortified with different concentrations of the cytokinin and auxin to evaluate their effects in this respect. The plant growth varied as the concentration of the growth regulators changed. The in vitro roots were successfully induced also.

2-The rooted plantlets were acclimatized  in magnolia trees , but the present study declared the necessary  for further studies to improve acclimatization to improve survival rate for the commmerical industry.