Abstract: A callogenesis protocol for Melaleuca alternifolia was developed in this study. The effects of different types of auxins, activated charcoal and combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin at various concentrations on the callus induction from leaf explants of M. alternifolia were investigated in order to determine the optimum callus induction and maintenance medium. The callus growth curve under the best callus maintenance medium was also studied. Calli were induced from leaf explants cultured on Murashige and Skoog (MS) medium incorporated with 1, 3 and 5 mg L-1 of 2,4-D, Indole-3-butyric Acid (IBA), Naphthaleneacetic acid (NAA) or picloram as well as MS medium with the combination of 3 mg L-1 2,4-D and 1, 2 or 3 mg L-1 kinetin, respectively. The calli developed from different types of plant growth regulators were varied in their morphological appearances. The maximum callogenesis response was obtained using the MS medium supplemented with 3 mg L-1 2,4-D. The control medium and the medium incorporated with NAA did not produce any callus. Addition of activated charcoal at 0.05 and 0.1% did not assist in the callogenic response, though it helped in removing the pigments and phenolic compound accumulations in the culture medium. The calli initiated from the best induction medium could not be maintained in the fresh new medium containing the same auxin composition. However, calli were successfully maintained in MS medium containing the combination of 3 mg L-1 2,4-D and 2 mg L-1 kinetin. The callus maintained in this treatment showed a sigmoid growth pattern and reached maximum the growth rate after three to five weeks upon cultivation.
Introduction
Melaleuca alternifolia, which commonly known as tea tree, is one of the well known medicinal herbs in the world. It is a small tree native to the northeast coastal region of New South Wales, Australia (Thal, 2005). The first recorded medicinal usage of M. alternifolia plant was by the Bundjalung Aborigines of northern South Wales (Carson and Riley, 1993). The leaves are the portion of the plant used medicinally (Thal, 2005). The essential oil of M. alternifolia, which is also known as tea tree oil has been used medicinally in Australia for more than 80 years, with uses relating primarily to its antimicrobial (Mondello et al., 2003; Carson et al., 2002) and anti-inflammatory (Koh et al., 2002) properties. M. alternifolia oil is obtained by steam distillation from the plant and contains about 100 components, which are mostly monoterpenes, sesquiterpenes and related alcohols (Hammer et al., 2004). The oil has endured fluctuating popularity in Australia and is currently a popular addition to numerous cosmetic and pharmaceutical products, which are available worldwide (Carson and Riley, 1993; Knight and Hausen, 1994).
The advent of plant tissue culture has enabled mass multiplication of plants. In the past, a maximum number of researches have been directed to develop tissue culture protocols for floriculture and fruit plants (Anonymous, 2005). Recently, research has also been carried out actively to develop tissue culture protocols for medicinal plants (Anonymous, 2005). Studies on the production of useful metabolite by plant cell culture have been carried out on an increasing scale since the end of the 1950's (Misawa, 1994). Medicinal plants such as M. alternifolia contain a variety of secondary metabolites which are useful medicines, food additives, perfumes, etc. For instance, M. alternifolia oil contains over 100 medicinally important compounds, of which α-pinene, terpene-4-ol, linalool and α-terpineol are lipophilic monoterpenes and the major active antimicrobial components of M. alternifolia oil (Carson and Riley, 1995; Kim et al., 1995; Raman et al., 1995). Previous studies have shown that some M. alternifolia oil components have varying degrees of activity against microorganisms (Carson and Riley, 1995). Therefore, further studies with pure components may be advantageous (Hammer et al., 1999). However, the concentration of each component in the oil can vary widely between trees inhabiting different geographical locations. For example, the leaf oil composition changes from South to North of its natural habitat, being 1,8-cineole rich in the South and high terpinen-4-ol, low 1,8-cineole in the North (Burfield and Hanger, 2000). Thus, plant tissue culture technique plays an important role in multiplying and producing M. alternifolia oil with desired standardized composition of secondary metabolites.
In view of the importance and advantages of plant tissue culture, as well as the lack of tissue culture studies on M. alternifolia, a tissue culture study was carried out in order to determine the effects of different plant growth regulators and activated charcoal at various concentrations on the induction and maintenance of callus from leaf explants.
Materials and Methods
Plant Materials
Young branches which were used as the source of leaf explants were
excised from the in vitro plantlet of M. alternifolia between
June to December 2005. The in vitro plantlet was cultured on the
MS medium containing 1 mg L-1 kinetin.
Culture Medium
MS medium (Murashige and Skoog, 1962) supplied with various concentrations
of plant growth regulators (2,4-D (Sigma, USA), NAA (Duchefa, Netherlands),
IBA (Duchefa, Netherlands) and picloram (Duchefa, Netherlands) at the
concentrations of 1, 3 and 5 mg L-1) in combination with 3%
(w/v) sucrose and 0.3% (w/v) gelrite (Duchefa, Nehterlands) were prepared
for callus induction. The pH of the medium was adjusted and maintained
at 5.7±0.1 using pH meter (Mettler Toledo) prior to autoclaving
(Hirayama, Japan) at 121°C for 15 min. To test the effect of activated
charcoal on callus induction, 0.05 and 0.1% (w/v) of activated charcoal
(Duchefa, Netherlands) was added into the MS medium supplemented with
1, 2 and 3 mg L-1 of 2,4-D prior to sterilization. For the
studies of the effect of auxin and cytokinin in combination on callus
induction, the plant growth regulators were replaced with 3 mg L-1
of 2,4-D in combination with 1, 2 or 3 mg L-1 of kinetin, respectively.
Callus Induction
Callus were initiated by culturing surface sterilized leaf explants
that were previously cut into squares in the size of approximately 0.5x0.5
cm on the MS medium containing either 2,4-D, NAA, IBA or picloram at the
concentrations of 1, 3 and 5 mg L-1 under sterile condition.
Callus induction percentage, day and degree of callus induction were determined
after 3 weeks of culture. In the study of the effect of activated charcoal
addition on callus induction, the leaf explants were cultured on MS medium
containing 1, 2, or 3 mg L-1 of 2,4-D with the addition of
0.05 or 0.1% (w/v) of activated charcoal on each of the medium, respectively.
Leaf explants also cultured on MS medium incorporated with 3 mg L-1
of 2,4-D and 1, 2, or 3 mg L-1 of kinetin in combination in
order to study the effect of combination of auxin and cytokinin on callus
induction. All cultures were incubated at 25±2°C under photoperiod
of 16/8 h with light intensity of 18 watts controlled by cool white fluorescent
light tubes (NEC). All the treatments were repeated three times with five
replicates for each experiment.
Callus Maintenance
Initiated callus were continuously subcultured to fresh maintenance
medium containing 3 mg L-1 of 2,4-D in combination with 2 mg
L-1 of kinetin for every 2 to 3 weeks interval. During subculture,
unhealthy brown callus were excluded from being transferred. All cultures
were incubated at 25±2°C under photoperiod of 16/8 h with light
intensity of 18 watts controlled by cool white fluorescent light tubes.
Results and Discussion
Callus Induction
Leaves were used as explant source because leaves are more amenable
to plant tissue culture as compared to other plant organs. Leaf explant
has been proven to be effective in inducing morphogenic response in tissue
culture studies of other plant species, such as Iris pumila (Jevremovic
and Radojevic, 2006) in callogenesis, Lycopersicon esculentum Mill.
(Sheeja et al., 2004) and potato (Yasmin et al., 2003) in
plantlet regeneration. The results obtained for callus induction from
leaf explant of M. alternifolia revealed that there was no sign
of callusing in the control medium. The explants cultured in the control
medium became unhealthy, turned to brown in colour in 3 weeks and eventually
died (Fig. 1a). These results were in accordance with
the results obtained by Jain et al. (2002), Li et al. (2002)
and Yasmin et al. (2003) whereby no callus were induced in Phlox
paniculata, Rosa hybrida, Rosa chinesis and potato,
respectively.
| Fig. 1: | Morphological differences observed between the calli initiated from different auxins throughout the culture period. (a) control after 3 weeks of culture; (b) 3 mg L-1 2,4-D after 3 weeks of culture; (c) 5 mg L-1 picloram after 2 weeks of culture and (d) 3 mg L-1 IBA after 4 weeks of culture. x showing the adventitious roots formed |
| Table 1: | Effects of MS medium supplemented with different auxins at various concentrations on the callus induction from leaf explant of M. alternifolia |
| aSD: Standard deviation; bRating of callus formed in size (cm); (-) absence of callus; (+) 0.1-0.2; (++) 0.2-0.3; (+++) 0.4-0.5; (++++) 0.6-0.7; (+++++) 0.8-0.9 | |
From all the four types of plant growth regulators tested, 2,4-D, IBA and picloram were successful in showing different degrees of callusing whereas NAA is the only growth regulator that failed to stimulate any callus formation (Table 1). Regardless of the concentrations of these auxins used, the calli were formed from cut edge of the explants and eventually covered the whole surface. Ruptured of the epidermis upon wounding of the explant has exposed the underlying tissue (Rahman and Punja, 2005) to the exogenous auxin, thus the exposed tissue was promoted to form callus in response to the effect of exogenous auxin supplemented in the MS medium. Similar results were reported by Rahman et al. (2004b) in the callus induction of Elaeocarpus robustus Roxb.
Different types of auxins have varying effects on plant growth and morphogenic response. Thus, the morphogenic potentialities of explant differ depending on the growth regulator supplements (Rahman et al., 2004a). This is shown in the present experiment whereby, it was observed that 2,4-D, IBA and picloram possess different intensities in inducing callogenic response. These observations were supported by Ramanayake and Wanniarachchi (2003) who reported that the different auxin treatments significantly affected the number of explants that induced callus. Apart from that, 2,4-D and picloram resulted in better callogenesis response as compared to IBA. Similar result was demonstrated in the tissue culture study of Muscari rameniacum whereby these two auxins (2,4-D and picloram) also showed better response than other auxin treatments (Suzuki and Nakano, 2001). The maximum percentage of callus formation (100%) was achieved in MS medium supplemented with 2,4-D at the concentrations of 1, 3 and 5 mg L-1, respectively. Generally, 2,4-D is known to function in stimulating cellular activity and the formation of morphogenic callus (Trifonova et al., 2001). According to Da Silva et al. (2005), 2,4-D is the main synthetic auxin used to induce the callogenesis, because one of its main characteristic is the capacity to efficiently stimulate the cell division in tissues of several plants. These statements are revealed in this study as the maximum callogenesis was exerted by the used of 3 mg L-1 of 2,4-D in MS medium. Treatment using 3 mg L-1 2,4-D also gave the best result in terms of day of callus formation. Thus, this concluded that MS medium incorporated with 2,4-D at 3 mg L-1 is the best callus induction medium for leaf explant of M. alternifolia. Likewise, these results were supported by Khatri et al. (2002) in the study of sugarcane, who reported that the best callus induction and proliferation was observed in the medium containing 3 mg L-1 of 2,4-D. However, the growth of initiated callus was unable to maintain in the induction medium and it turned to brown in colour after 4 weeks of culture. The brown callus indicated stress callus and exhibited reduced morphogenic response (Shibli et al., 2001). The browning of callus could be due to the exposure of stress situation exerted by the detrimental effect of 2,4-D, which is also act as auxinic herbicide (Mithila and Hall, 2005).
Picloram showed relatively high callogenic capacity compared to IBA. In fact, nearly 100% of the explants inoculated induced calli in 1 to 5 mg L-1 of picloram. Small proportions of the explants that did not initiate callus were unhealthy and died as a result of pigments or phenolic accumulations. Similar result was demonstrated by Cardoza and Souza (2002) whereby 0.5 and 1.0 mg L-1 of picloram gave the highest amount of callus in Anarcardium occidentale L. Nevertheless, the calli initiated with picloram could not be maintained as it turned brown and necrotic within 2 weeks of culture, most probably due to phenolic oxidations. This observation was in accordance with the tissue culture study of Calamus subinermis and Calamus merrillii in which Goh et al. (2001) indicated that the calli produced from the treatment of picloram eventually turned to brown in colour and became necrotic after several weeks of culture. Goh et al. (2001) also proposed that these observations were most likely due to phenolic oxidations. In fact, it has been suggested that the accumulation of pigments or phenolic compounds is a response to either microbial infection and/or physiological stress such as wounding of tissues, which is a part of the defense mechanisms in plant whereby phenolic production serves to limits microbial invasion of cells (Rahman and Punja, 2005). As shown in this tissue culture study, wounding of the leaf explants promoted the damaged tissues or cells to released pigments or phenolic compounds in respond to the stress condition. Eventually, brown or dark colour products were formed as a result of oxidation reaction of these compounds. According to Alemanno et al. (2003), one of the factors often considered as a component of in vitro recalcitrance is a high phenolic content and oxidation of these compound and the oxidation could be a limiting factor preventing proper tissue multiplication and maintenance. Thus, in the present experiment, M. alternifolia was proved to be a recalcitrant plant species as some of the cultured explants were unable to be induced into callus and became unhealthy and eventually died due to the secretions and oxidation reaction of some pigments or phenolic compounds in the cultured medium.
Since picloram is an auxinic herbicide (Mithila and Hall, 2005), in the present experiment, the browning of callus in the treatment of picloram could also be due to the phytotoxicity of picloram. The degree of browning was also fastened as the concentration of picloram applied increased, most likely due to the adverse effects of the high level of plant growth regulators whereby high concentration of plant growth regulator can act at molecular level and exert detrimental effects on the plant tissue (Aslam et al., 2005). In addition, the increased concentration of picloram lead to more intense deleterious herbicide effect on the callus and therefore accelerate the browning and necrosis process of the callus. Similar result in the callus culture of other plant species such as Olea europea L. and cotton further confirmed this observation. Shibli et al. (2001) reported that high concentration of 2,4-D (>2.21 mg L-1) resulted in the development of brown colour callus in the tissue culture study of Olea europea L. whilst Aslam et al. (2005) reported that higher BA level produced detrimental effect on callus initiation in cotton.
IBA showed relatively slower callus induction response compared to 2,4-D and picloram. Similarly, lower percentage of callus formation was also observed in the IBA treatment. The highest percentage of callus induction obtained within the IBA treatment was only 50%, which was induced in the concentration of 5 mg L-1 of IBA (Table 1). Callusing was observed after 15 days of culture. Apart from slower and lower percentage of callus formation compared to the treatments of 2,4-D and picloram, the size of the calli induced in IBA were also relatively smaller (approximately 0.1 to 0.2 cm in length). In the IBA treatment, adventitious roots were also developed in the explants cultured in IBA at the concentrations of 1, 3 and 5 mg L-1 (Fig. 1d). Likewise in other plant species such as Elaeocarpus robustus Roxb. and Morus latifolia, rooting of explants in the present of IBA was observed (Rahman et al., 2004b; Lu, 2002).
There was no sign of callusing in the MS medium incorporated with NAA as a source of auxin. This result is contradicted with the results obtained in callogenesis of other plant species, such as potato (Yasmin et al., 2003) and Macrotyloma uniflorum (Mohamed et al., 2005) whereby NAA was found to be effectively stimulated callogenesis response in the particular plant species. This varied effect of NAA might be due to the fact that the effect of plant growth regulator is plant specific, thus different plant species respond differently to the same type of plant growth regulator.
Generally, callus cells dedifferentiated without uniformity and shows genetic, structural and physiological variability (Mukhopadhyay et al., 2005). As presented in this study, calli with different appearance were initiated. According to Rossi-Hassani and Zryd (1995), growth regulators affected callus texture and morphology. Thus, the calli developed from different auxins exhibited different texture and morphological appearance. Calli induced from 2,4-D were greenish yellow in colour, nodular and friable (Fig. 1b) whilst calli initiated using picloram were yellowish brown and more friable (Fig. 1c). In the IBA treatment, the calli developed were brownish and compact (Fig. 1d).
Effects of Activated Charcoal on Callus Induction
Activated charcoal is a compound that adsorbs toxic compounds released
to the culture medium from the explants or from the agar, sucrose and
salts. Its presence in the medium favours the growth and further development
of the embryos (Ricci et al., 2002). Nevertheless, activated charcoal
adsorbs not only inhibitory substances accumulating in the cultural medium
but also cytokinins and auxins present in the medium (Han et al.,
2005).
In the present experiment, the addition of 0.05 and 0.1% of activated charcoal effectively eliminated the accumulation of pigments and phenolic compounds in the medium (Fig. 2). Thus, preventing the inhibitory effect of these components on the growth and development of the explants cultured. Similar observation was demonstrated by Asao et al. (2003) whereby it was found that the addition of activated charcoal has successfully recovered the vegetative growth and corm yield of taro plants, suggested that this outcome was due to the absorption of the exuded allelochemicals by activated charcoal. However, in this study, there was no sign of callus initiation being observed on the cultured explants upon the addition of activated charcoal. This suggested that the treatments of activated charcoal inhibited callogenesis response in M. alternifolia, ascribing to the absorption of growth regulator (2,4-D) by activated charcoal in the medium. These results were supported by Lu (2002) who reported similar inhibitory impact of activated charcoal on callus formation in Morus latifolia.
| Fig. 2: | The effect of addition of different concentrations of activated charcoal on callus induction form leaf explant of M. alternifolia after 2 weeks of culture in MS medium containing 1 mg L-1 of 2,4-D. (a) Medium added with 0.1% of activated charcoal; (b) without activated charcoal. x and y showing the excretion of pigments or phenolic compounds in the explant and medium |
Similar observation was also reported by Agarwal et al. (2004) who stated that the activated charcoal adsorbed a number of compounds including auxins and culture metabolites, resulting in the inhibition of embryo maturation in Morus alba. This result is in contrast with Lilium in which activated charcoal showed positive effects on bublet growth (Han et al., 2005). Thus, it was made clear that the incorporation of activated charcoal in the tissue culture medium may either have a beneficial or adverse effect on the growth and morphogenic response of plant tissue, depending on the species and tissue used (Lu, 2002).
Effects of Combination of Auxin and Cytokinin on Callus Induction
In plant tissue culture, most tissues require a combination of specific
plant growth regulators to produce the appropriate growth response of
the tissues (Trigiano and Gray, 2000). This is revealed in the present
tissue culture study whereby the combination of 2,4-D and kinetin has
successfully stimulated callogenesis response. Similar with the results
in the single auxin treatment, the combinations of auxin and cytokinin
at different concentrations tested resulted in maximum callogenesis response.
This observation was supported by the tissue culture study of Foeniculum
vulgare (Anzidei et al., 2000) and Valeriana edulis sp.
(Castillo et al., 2000) whereby both the treatment of 2,4-D alone
or in combination with kinetin were effective in showing relatively higher
percentage of callogenesis response. It was observed that the degree of
callusing was the highest in the MS medium incorporated with 3 mg L-1
of 2,4-D and 2 mg L-1 of kinetin. Besides, the calli initiated
from MS medium incorporated with the combination of 3 mg L-1
2,4-D + 2 mg L-1 kinetin showed the highest growth with the
increment of 0.22 cm in callus size after four weeks of induction, as
compared to 0.13 cm in MS medium containing 3 mg L-1 2,4-D
+ 1 mg L-1 kinetin and 0.2 cm in MS medium containing 3 mg
L-1 2,4-D + 3 mg L-1 kinetin. Likewise in the callus
induction of Cephaelis ipecacuanha, the treatment with the combination
of relatively higher concentration of 2,4-D and lower combination of kinetin
promoted rapid callus growth from the leaf explant of Cephaelis ipecacuanha
(Rout et al., 2000).
Callus Maintenance
The leaf derived calli of M. alternifolia that were subcultured
onto fresh induction medium, MS medium containing 3 mg L-1
2,4-D showed brown coloration, turned necrotic and unable to be maintained
(Fig. 3b). Therefore, media trials with different combinations
of 2,4-D and kinetin were conducted. Improved callus growth was observed
in all the combinations tested, probably due to presence of kinetin that
aided in prolonging the growth of callus by enhancing the cell division
in the callus tissue (Aslam et al., 2005). Apart from that, it
was discovered that the calli were best maintained in the combination
of 3 mg L-1 of 2,4-D and 2 mg L-1 of kinetin as
they exhibited the highest callus growth rate and the calli produced were
morphogically more compact and greenish in colour (Fig.
3a). These observations suggested that the combination of 2,4-D and
kinetin effectively enhanced growth and maintenance of leaf derived calli
of M. alternifolia. Similar auxin and cytokinin combinations proved
to be more effective in the maintenance of callus growth of other plant
species, such as Canavalia brasiliensis (Da Silva et al.,
2005) and Elaeocarpus robustus Roxb. with lower level of 2,4-D
(Rahman et al., 2004b). The calli which were initially more friable
and yellowish brown in colour turned to green in colour and more compact
upon subsequent subculturing onto the fresh maintenance medium, which
was MS medium incorporated with the combination of 3 mg L-1
of 2,4-D and 2 mg L-1 of kinetin. This suggested that the calli
growth and development were improved in this medium. Such morphological
changes that take place during callus maintenance were also observed in
Phlox paniculata (Jain et al., 2002) and Anacardium occidentale
L. (Cardoza and Souza, 2002).
| Fig. 3: | Morphological differences between callus maintained on the same induction medium and that maintained on MS medium containing combination of auxin and cytokinin. (a) Callus maintained on MS medium with 3 mg L-1 of 2,4-D and 2 mg L-1 of kinetin in combination; (b) Callus maintained on MS medium with 3 mg L-1 of 2,4-D |
As observed in the present study, it was suggested that many plant species require supplementary of cytokinin along with auxin for optimum response and in some cases to prevent necrosis of callus (Roy and Banerjee, 2003; Wang et al., 2004). This requirement for exogenous cytokinin could be related to the maintenance of a proper balance between auxin and cytokinin, which act synergistically to regulate cell division (Roy and Banerjee, 2003).
Since the compact calli have great organogenic potential, it is suggested that the study of in vitro regeneration from the leaf derived callus can be carried out to facilitate mass propagation of M. alternifolia, since high multiplication rate could be achieved using micropropagation rather than traditional vegetative propagation methods. In addition, the studies of somatic embryogenesis can also be conducted in developing an efficient embryogenic culture system that is useful for the purposes of genetic transformation and biolistic gene transfer for in vitro plant improvement. Thus, this offers an efficient mean of rapid large-scale propagation of the transgenic plants and superior genotypes of M. alternifolia which subsequently serve as a source of high quality secondary products and medicinal raw materials. Apart from that, in view of the enormous amount as well as high commercial value of the secondary metabolites contain in the essential oil of M. alternifolia, essential oil extraction from the callus cultures or in vitro plantlet of superior genotypes of M. alternifolia can also be carried out for the production of high quality essential oil as well as to standardize the yield of essential oil.
Acknowledgments
This research was supported by Universiti Tunku Abdul Rahman (UTAR), Malaysia.