IN VITRO PROPAGATION OF Eucalyptus citriodora PLANT

This work was carried out in Plant Tissue Culture Laboratory of Prof. Dr. Abd El-Fatah Helmy Belal, Faculty of Environmental Agricultural Sciences, Arish University, North Sinai, Egypt during the period from 2016 to 2018. This study was conducted to study the effect of medium type, explant source and growth regulator type and concentration on micropropagation of E. citriodora plant which is grown in Sinai Peninsula. Results showed that full strength of Murashige and Skoog's medium (MS) was the most suitable medium for seed germination and shoot growth. Meanwhile, addition of BA at 1.00 mg l was effective for improving shoot growth and development. Also, multiplying of shoots was enhanced by using nodal cutting explants cultured on MS medium supplemented with 1.00 mg l BA plus 1.00 mg l NAA. On the other hand, shoot rooting was achieved by using MS medium supplemented with 2.50 mg l IBA. It is worth to mention that obtained plantlets were successfully acclimatized (80% survivability) in a combination of peatmoss, vermiculite and washed sand or peatmoss and vermiculite at equal volumes.


INTRODUCTION
The Eucalypts genus is a member of the Myrtaceae family, it include three genera; Eucalyptus, Corymbia and Angophora (Pandey, 1987;Trueman and Richardson, 2007). Eucalyptus citriodora is a large, evergreen tree, 24-40 m (max. 50 m) in height; leaves are strongly lemon-scented and fruits are urnshaped (Orwa et al., 2009). The economic importance of E. citriodora plant was producing pulp, producing wood for timber, oil distillation (which used in perfumery, antiseptic for relieving coughs and colds, insect repellents especially against mosquitoes), planted for reforestation and for ornamental garden (Hill and Johnson, 1995). The genus of Eucalyptus is generally propagated by seeds at present; however seedlings did not exceed 50% successful establishment and self-incompatibility found between each of them. Whilst it is very difficult or/and failure to propagate by vegetative propagation such as budding, grafting and layering due to problem of incompatibility between scion and rootstocks (Girouard, 1974;Zobel, 1993;Verma et al., 2013;Bryant and Trueman, 2015).
In order to increase the efficiency of in vitro techniques, tissue cultures have made use of purified chemicals and sophisticated physical facilities where by the temperature, humidity, lighting conditions and aeration are controlled. It is also important to improve total production of ornamental, medicinal and aromatic plants or increasing the yield per feddan by using tissue cultural techniques (Nizar, 2001). Also, Ho et al. (1998)  Pasha and Irfan (2011) reported that the highest percentage of bud break and initiation of Eucalyptus citriodora shoots was reported in the medium containing benzyladenine and kinetin at 0.5-0.2 mg l -1 after 10-15 days of culture. It is known that the function of rooting stage is to prepare the plantlets for transplanting and establishment outside the artificial closed environment of culture vessels (Murashige, 1974). Thus, Koriesh et al. (2003) found that Eucalyptus citriodora shoots rooted when using full strength MS medium fortified with 1 or 2 mg l -1 IBA. While, Rahman et al. (2013) mentioned that the highest percentage of rooting of the shoots of Paulownia tomentosa occurred with addition of IBA (0.5 mg l -1 ) compared with culture medium without growth regulators. Meanwhile, acclimatization stage is considered one of the very important stages for in vitro micropropagated plants which affect plant survival. Also, they recorded the maximum percentage of acclimatized plantlets survival with the mixture of compost, soil and sand (1:1:1, v/v) for Paulownia tomentosa Steud.
Thus, the main objective of this experiment was to study the effects of in vitro culture media type and explant source as well as growth regulator type and concentration on micropropagation of Eucalyptus citriodora trees which grown in Sinai Peninsula.

MATERIALS AND METHODS
This study was carried out at Prof. Dr. Abd El-Fatah H. Belal Tissue Culture Laboratory, Faculty of Environmental Agricultural Sciences, Arish University, North Sinai, Egypt, during the period from 2016 to 2018 to study the effects of in vitro culture media type and explant source as well as growth regulator type and concentration on micropropagation of Eucalyptus citriodora trees which grown in Sinai Peninsula.

Plant material
Eucalyptus citriodora L. seeds were obtained from Gene-Bank at Desert Research Center (DRC), El-Sheikh Zuwyed, North Sinai, Egytpt.

Seeds sterilization
Seeds were shaked with a few drops of liquid soap for 5 minutes then rinsed under running tap water for 60 minutes. After that seeds were soaked for 30 sec. in 70% ethyl alcohol followed by soaking for 5 minutes in 15% Clorox (containing 5.25% sodium hypochlorite). Seeds were thoroughly rinsed three times with sterile distilled water after each previous step.

Recorded data
After 28 days from inoculation date, seed germination percentage (SGP), seedling vigor index (SVI), shoot length (cm) and number of leaves/shoot

Multiplication stage
This stage aimed to increase the number of shoots, so that shoots obtained from establishment stage was used as explant source during multiplication experiments to study the following factors:

Effect of explant source and media type
Shoot-tips or one-node cuttings of germinated plants of E. citriodora were cultured on different media type i.e.; Murashige and Skoog (1962), Woody Plant Media (Llyod and McCown, 1980) and Schenk and Hildebrandt (Schenk and Hildebrandt, 1972) to select the best explants source which encouraged the highest growth development and best medium type that induce the highest explant development.

Effect of explant source and cytokinin type
Aseptic seedlings of 4 weeks old of E. citriodora were used. Every shoot was divided into two explant parts; shoot-tip and nodal cutting. Each explant was about 1-1.5 cm length. These explants were cultured on MS media which supplemented with three cytokinin types; Kinetin (Kin), 6-benzyladenine (BA) and 2-isopentenyl adenine (2iP). Each one was studied at the rate of 1.00 mg l -1 compared with free MS medium (control) to detect the best cytokinin type which could induce the highest multiplication.

Effect of explant source and different BA and NAA concentrations
Shoot-tips or one-node cuttings of E. citriodora were cultured on MS media which supplemented with two concentrations of benzyladenine (0.50 and 1.00 mg l -1 ) in combination with four (concentrations 0.10, 0.50, 1.00 and 1.50 mg l -1 ) of αnaphthaleneacetic acid (NAA). Also, free MS medium (without growth regulators) was tested. Each treatment consisted of 12 jars and each one contained 30 ml of medium. This study conducted to investigate the most suitable combination between Kin and NAA that induce the highest multiplication rate.

Recorded data
Shoot length (cm), number of leaves/shoot, number of shoots/explant and number of leaves/shoot were recorded after 30 days from inoculation date.

Rooting stage
The proliferated shoots obtained from the multiplication treatment (nodal cutting on medium supplemented with BA and NAA at 1.0 mg/l) were used as explant source and cultured on Murashige and Skoog (MS, 1962) supplemented with 100 mg l -1 myoinositol, 30.0 g l -1 sucrose and 7.00 g l -1 agar. These shoots were grown on MS medium free from growth regulators for 4 weeks before using as explant source for the following experiment to eliminate any residual effect of PGRs that might inhibit or reduce rooting.

Effect of auxins type
Shoots (3-4 cm length) were excised from the proliferated shoots and cultured on MS media supplemented with 1.00 mg l -1 of indole-3-butyric acid (IBA), α-naphthaleneacetic acid NAA or indole acetic acid (IAA) to determine the best auxin type that could enhance the best root formation in E. citriodora.

Effect of IBA concentration
Shoots (3-4 cm length) were cultured on MS medium supplemented with different concentrations (0.00, 0.50, 1.00, 1.50 and 2.00 mg l -1 ) of IBA to investigate the suitable concentration which could encourage the highest root formation rate.

Recorded data
Shoot length (cm), number of leaves, number of shoots/shoot, number of leaves/shoot, number of main roots/ Shoot and root length (cm) were recorded after 30 days from inoculation date.

Acclimatization stage
Rooted plantlets (about 8-9 cm length) of E. citriodora were acclimatized by transferring them to plastic pots (9 x 7 cm) containing peatmoss, vermiculite and washed sand or peatmoss and vermiculite at equal volumes. After 30 days survival percentage was recorded.

Statistical Analysis
Each experiment set up in a completely randomized design (CRD) with four replicates and each replicate consisted of three jars each one contained four explants. Data were statistical analyzed with analysis of variance (ANOVA) procedure using SAS statistical software package (SAS, 2004). Differences between means were compared by using Duncan's multiple range test (Duncan, 1955) at 0.05 level of probability. Table 1 show that the highest germination percentages were obtained when seeds were cultured on full, half or quarter MS strengths medium without significant differences among these treatments, while tenth strength of MS media achieved the lowest germination percentage, seedling height, number of leaves and the seedling vigor index (72.50 %, 2.86 cm, 5.87 and 2.07, respectively). There were a little variations among different medium strengths (especially full, half and quarter strengths) concerning seedling length, number of leaves/shoot and seedling vigor index. This result was in agreement with that of on Dipteyx alata where they concluded that a 25% concentration of MS salts was the best option for the in vitro establishment of these plants.

Effect of explants and media type
Data presented in Table 2 clear that the tallest shoots were recorded when MS medium was used regardless explant source (nodal cutting or shoot tip). While, the maximum number of leaves/shoot was gained when nodal cuttings or shoot tips were cultured on MS medium or as nodal cutting was cultured on SH medium without significant differences among these treatments. There were a little variations among different investigated treatments concerning No. of shoots/explant.  Table 3 show that the highest value of shoot length (1.52 cm) was belonged to nodal cutting which cultured on MS medium supplemented with 1 mg l -1 BA. While, the lowest value of shoot length was belonged to shoot tip explant which inoculated in MS medium enriched with 1 mg l -1 2ip. Concerning

Effect of explant type and different BA and NAA concentrations
Data in Table 4 show that the highest shoot length (cm) and number of leaves /shoot (2.45 cm and 2.45 leaves/shoot, respectively) were recorded when nodal cutting was cultured on medium supplemented with BA and NAA at 1.0 mg/l for each one. Also, using the same explant source combined with 0.5 mg/l BA and 1.0 mg/l NAA gave almost the same number of leaves/shoot. There were no wide variations among different tested treatments concerning number of shoots/explant. This result was in accordance with those obtained by Sharma and Kalia (

Effect of auxin type
Data presented in Table 5 indicate that growth regulator type had a significant effect on shoot growth and rooting, since shoot length the highest shoot length, number of shoots/explant, number of roots/shoot and root length of E. citriodora were gained when IBA was used. On the other side, the maximum number of leaves/shoot was recorded as IBA at 1.00 mg l -1 was used. It is worth to mention that there were no significant differences among different auxin types concerning number of roots/shoot. These results are in a harmony with those obtained by De Fossard et al. (1973) and Das and Mitra (1990) since they indicated that culturing the nodes of Eucalyptus in nutrient media supplemented with IBA enhanced the roots development and formation. On the same line, Koriesh et al. (2003) indicated that the root number and root length were increased by using medium supplemented with IBA compared with non-treated medium (control).

Effect of IBA concentration
Results presented in Figures 1 and 2 as well as Photo 1 show that addition of IBA at any applied concentration significantly improved shoot and root growth. The highest value of number of shoots was gained with the highest concentration of IBA. No. of leaves/shoot did not significantly affect by increasing IBA concentration in the range of 0.5 to 2.0 mg/l while higher concentration (2.5 mg/l) had depressive effect in this regard. It is obvious that high concentrations (1.5, 2.0 and 2.5 mg/l) were more effective than low concentration for increasing No.