An Efficient Protocol for Callus Induction in Aquilaria malaccensis Lam. Using Leaf Explants at Varied Concentrations of Sucrose

Aquilaria malaccensis Lam. (Family: Thymelaeaceae), commonly known as agarwood, eagle wood or Gaharu is a commercially important tree species of northeast India. The Aquilaria tree is categorised as critically endangered in Ind ia. This species is being continuously exploited due to its precious heartwood which is the source of expensive agar oil used in the production of high grade perfumes as well as in trad itional medicines. The main aim of this study was to establish a speedy callus production protocol from Aquilaria malaccensis which may serve as an important option for direct extraction of agar o il. For this purpose, large scale p roduction of callus tissue is needed. An efficient callus regeneration protocol was established through leaf pro liferation in Aquilaria malaccensis Lam. using Murashige and Skoog medium supplemented with least amount of plant growth hormones (2,4 D, NAA, Kinetin & BAP) at varied concentrations of sucrose (2-5%). Callus could be initiated in all the treatments of hormones and sugar levels; however, the best callus growth was obtained in the MS medium supplemented with BAP (0.5 mg/ l) + NAA (3mg/l) g iving the h ighest fresh (7.368g) and dry cell b iomass (2.170g) at the optimum sucrose concentration (4%) after 45-60 days of incubation.


Introduction
In Ind ia, th e med iu m s ized ev ergreen tree Aquilaria malaccensis Lam. (syn. Aquilaria agallocha Ro xb. of family Thymelaeaceae) co mmon ly known as agarwood, eagle wood or Gaharu is confined to Eastern Himalayas up to an altitude of 1000m. The term 'Ag arwood ' refers to its res inous heartwood which turns aro matic and highly valuable as a result of infections by a few endophytic fungi [1]. There are fifteen species in the genus Aquilaria of which eight are known to produce agarwood [2]. A. malaccensisis chiefly distributed in 10 countries viz. Bangladesh, Bhutan, India, Indonesia, Iran, Malaysia, Myanmar, Philippines, Singapore and Thailand [3]. In India, it is occurring mainly in Arunachal Pradesh, Assam, M izoram, Manipur, Nagaland, Sikkim and West Bengal [4]. A. malaccensis is known to be one o f the mo s t imp o rtant s pecies o f co mmerce and v alued fo r production of its impregnated resinous heart wood that gives frag rance [5]. The in fected p arts o f ag arwood serve as imp o rtant raw material in th e p rodu ctio n o f incense, perfu mes and traditional med icines [6]. The agar wood oil or aloe wood oil, known in the east as 'agar attar' is obtained by distilling selected parts of the infected wood of Aquilaria sp. which has unique fragrance and high export value [7]. The agar oil traders have to sacrifice whole t ree as its heart wood serves as raw materials for oil distillation. Many uninfected or less infected trees are also been destroyed by them in search of agarwood. Due to such exp loitation, this tree species is now rarely found in wild habitat and considered as critically endangered in India. Consequently, it is included in IUCN red data list of the year 2011 as vulnerable and at the verge of ext inction fro m the natural forests [8]; hence there is an urgent need to apply modern technologies for the conservation and existence of this particular species as well as for preservation of its germplasm. The application of biotechnological methods such as plant tissue culture technique may be the best alternative for the above purposes. Plant t issue culture is also important for the species which has very short dormancy period and dependent on any particular reproductive period as in case of tradit ional method of sexual reproduction. The A. malaccensis La m. also have restricted period of seed viability [9]. The plant is generally propagated through seeds who germinate readily after maturity; however their rate of germination sharply reduces with the increase in the period of storage/decrease in its mo isture contents. Fresh seeds obtained by splitting open the capsule showed a short dormancy period ranging fro m 1 week to 4 weeks only. The maximu m germination (90%) takes place within 20 days in case of fresh seeds and 7 days for stored seeds with very less germination percentage. Seed weight has a positive influence in germination, plant size, root length and ultimately in yearly plant establishment [9]. The in-vitro propagated medicinal p lants furnish a ready source of uniform, sterile and co mpatible p lant material for mass mu ltiplication and help in germplas m conservation of rare, endangered and threatened medicinal p lants. Taking these factors into consideration, techniques of plant tissue culture were adopted by many scientists for propagation of Aquilaria seedlings and to supplement the conventional methods of regeneration [10], [11]. By using tissue culture raised agarwood plantlets, growers may obtain mo re oleoresin than those plantlets grown fro m random seeds. The uniform growth with t issue culture plantlets may make the process of inducing oleoresin format ion easier and volu me of oleoresin more predictable. However; the proper and long-term monitoring of these seedlings is essential fo r their adaptation in natural conditions and for afforestation and plantation programmes fo r the socio-economic benefits of local populace.
Agar oil production from callus through plant tissue culture is another option wh ich may be co mmercially exploited. In-vitro production of agar oil fro m callus culture of A. agallocha Roxb. and analysis of its chemical constituents by GC/MS have already been done successfully [12]. Chemical analysis of the oil produced from callus however showed some variat ions in the quality o f the oil, when compared with the oil obtained by hydro distillat ion of fungal infected agarwood. GC/MS analysis has shown the presence of about 32 different compounds comprising of furano-monoterpenoids, acids, alcohols and aldehydes, out of which about 15 co mpounds could be identified. About 30% of the identified co mpounds showed similarity with the original agarwood oil. More such studies are needed to characterise and compare oil production capacity of calli and respective oil quality. The effect of fungal imp regnation on callus and agar oil production may be another field of research which is required. Therefore, the present studies initially aimed to standardize an efficient, rapid and less expensive protocol for the production of callus tissue in vitro and study of salient features and growth parameters of callus tissues of A. malaccensis La m., so obtained that may be utilised for essential oil production in future.

Materials and Methods
Leaf tissues from 3-6 month old seedlings of A. malaccensis Lam. were used as explants for callus induction. Exp lants were surface sterilized as per modified method of De [13] with a few drops of Tween-20 for 15minute followed by thorough washing (3-4 t imes) with distilled water followed by with sterile distilled water. Further steps were done under complete aseptic conditions using a laminar air flow cabinet. The exp lants were then treated with 70% ethanol for 1 minute, with 5-10% (v/v) sodium hypochlorite solution for 15 minutes and finally washed with sterile distilled water for 3-4 times to remove the traces of chemicals. Different hormonal co mb inations were tested at their varying concentrations ranging from 0-10mg/l 2,4 -Dichlorophenoxyacetic acid (2,4-D,) + 0-3mg/ l Kinetin (Kn), 0.5-4mg/l 6-benzy ladenine purine (BAP) + 0.5-4mg/l α-naphthalene acetic acid (NAA) and 0-10mg/l 2,4 -Dich lorophenoxyacetic acid (2,4-D) + 0.5-4mg/l 6-benzy ladenine purine (BAP) fo r callus induction in A. malaccensis (Table 1). One concentration fro m each combination that had given the highest bio mass of callus was selected for comparative studies. The surface sterilized explants were placed on MS mediu m [14] supplemented with different concentrations & co mbinations of p lant growth regulators at varying sucrose concentrations (2-5% w/v) to check their effect on callus induction, growth and development [12]. The three combinat ions i.e. 2,4-D (2 mg/l) + Kn (0.1mg/l), BAP (0.5 mg/l) + NAA (3mg/l) and 2, 4-D (2 mg/l) + BAP (0.5mg/l) were tested and represented as combination A, co mbination B and combination C respectively. The pH of the mediu m was adjusted at 5.7 to 5.8 with the help of NaOH or HCl prior to adding 0.8% bacto agar and autoclaved at 121ºC for 20 minutes. All the cultures were inoculated in 5 replicates and incubated at 25º± 2ºC in complete dark condition. A set of flasks were also kept in light illu mination (light intensity ranging fro m 20-40µE/ m 2 / S 1 ) to check the effect of light on callus growth. The percentage of callus bio mass on fresh weight (FW) and dry weight (DW) basis was recorded after the period of 15-30, 45-60 and 75-90 days respectively. Dry weights were taken to check the mo isture contents (data not shown) in young and mature callus tissues. The morphological and microscopic features of Aquilaria callus were also studied as per the method prescribed by Deka [15].

Results and Discussion
In this paper, the emphasis was given on induction of callus in medicinally impo rtant tree species A. malaccensis Lam. using leaf exp lants. The induction and growth characteristics of Aquilaria callus from leaves, shoot tips and stem as explants were also reported earlier by Talukdar and Ahmed [16]. They have also obtained best callus using leaves as explants; hence, in the present studies, leaves were used for callus induction in this important tree species; however, present protocol was standardized at comparatively very reduced concentration of growth hormones. The results of our studies indicate that the optimu m incubation period required is about 45-60 days in contrast to Talukdar and Ahmed [16] who obtained maximu m callus in 45 days. This may be due to sub-culturing of callus in fresh med iu m by the investment of quite higher concentration of plant growth hormones. The hormone comb ination B i. e. BAP + NAA at Lam. Using Leaf Explants at Varied Concentrations of Sucrose the concentration of 0.5 mg/l & 3 mg/l respectively was found to be the best for fast callus induction and further proliferation in the test plant species (Table1). The MS med iu m supplemented with hormonal co mb ination 0.1mg/l NAA+ 0.5 mg/ l BA was also found suitable and gave the best result in the formation of protocorm-like bodies (PLB) obtained from shoot tips of Cymbidium species [17]. We as well recorded the 4% concentration of sucrose as most favourable in M S med iu m as standardized earlier by Talukdar and Ahmed [16] however; callus biomass at 3% sucrose concentration was also considerably high some times highest (7.231g, 6.853g & 7.210g in co mbination A. B &C respectively) especially after 45-60 days of incubation. The dry cell bio mass of callus was found to be proportional to their fresh cell b io mass (Table1). In general, the callus growth declined in all treat ments except in case of hormonal combination C, where an increase in fresh to dry cell biomass ratio was noticed.
We recorded the highest callus bio mass in the M S mediu m supplemented with hormone combination B as 7.457g FW and 2.302g DW (Table1) at optimu m concentration of sucrose (4%) after about 45-60 days of incubation. The lowest callus cell bio mass was observed in hormones combination C at 2% sucrose concentration as 0.931g FW and 0.229g DW callus cell b io mass after same duration of incubation.
In hormone co mbination A, the maximu m callus bio mass was obtained (7.368g FW) in 45-60 days old culture at 4% sucrose concentration whereas the lowest was recorded with 2% sucrose (1.661g FW) concentration. In co mbination B, the maximu m callus bio mass (7.457g) was obtained in 4% sucrose and the lowest was observed in 2% sucrose concentration (0.493g) after 45-60 days of incubation. Callus growth in hormone co mbination C depicted that 30% sucrose concentration supported the highest callus cell bio mass (7.210g ) and the lowest cell bio mass measured in case of 2% sucrose concentration (0.931g ) after 45-60 days of incubation.
In 75-90 days incubation time, it was interesting to note that there was decrease in fresh cell bio mass however, the dry cell bio mass increased. The reason might be due to the fact that lignification in callus tissues might have taken place as a result of cyto-differentiat ion in older callus tissues and made them harder (Figure1d). It was also evident by the changes in the colour and appearance of 75-90 days old callus which looked dull and darker.
Sucrose had various effects on callus initiation and growth ( Figure 3). It is the most common source of carbohydrate needed in callus induction and regeneration of plants [18]. The induction of callus occurred comparat ively earlier and its growth was recorded faster in case of media treated with 4% sucrose irrespective of hormonal treat ments up to 45-60 days of incubation ( Figure 3C). The b io mass of callus was universally proportional to the concentration of sucrose up to 4% and declined beyond it. Fro m the present investigations, it was revealed that prolonged culturing period of callus has direct impact on its growth as weight of the callus declined sharply after 60 days of incubation. In contrast to the present findings, Talukdar and Ahmed [16] reported the highest growth of the callus on MS med iu m supplemented with quite higher amount of gro wth hormones (6mg/l 2, 4 D + 2mg/l Kn) within 30 -45 days of culture however concentration of sucrose was optimized as 4% which support our findings. In another experiment, the highest growth of Aquilaria agallocha callus (547.25%) was obtained using 3% fructose as source of carbohydrate by keeping the hormonal treat ment constant through leaf explants after 45 days of culture [19]; however, the maltose (60 g/l) d id not supported callusing and showed the least growth rate (212.76%) in fresh cell biomass. Table 1. Effects of different growth hormones on biomass productionof A. malaccensisLam. in MS medium supplemented with different concentrations of sucrose, A represents 2, 4-D (2 mg/l) + Kn (0.1mg/l); B represents BAP (0.5 mg/l) + NAA (3mg/l); C represents 2, 4-D (2 mg/l) + BAP (0.5mg/l) Fro m the present experiment, it can be concluded that after 45-60 days of incubation, hormonal comb ination B and 4% sucrose were best options for rapid callus growth in A. malaccensis Lam. In all the treat ments, 2% sucrose showed least growth (0.931g m) as co mpared to other sucrose concentrations (Figure3A). In 15-30 days old callus, the highest growth was recorded in comb ination B (2.390g FW) followed by combination A (1.281g FW) and the lowest growth (0.259g FW) was observed in combination C in 4% sucrose ( Figure 3C); but in 75-90 days old cu lture, callus in all t reat ments were declined except in hormone co mbination A with 3% (Figure3B) sucrose and combination C with 2% sucrose (Figure3A) where the negligible amount of callus were increased. No clarification could be drawn for this. At 5% sucrose concentration, reduced growth of callus was recorded however; the highest growth was noted in the hormone co mbination A (Figure3D). Callus tissues of A. malaccensis which were grown and maintained under dark beco me whitish or creamish yellow in colour (Figure 1 a-c). They usually turned green when transferred under light illu minations (intensity 20-40µE/ m 2 / S 1 ) due to development of chloroplastids. Aceto-carmine staining of young and matured callus tissues revealed the presence of embryogenic cells [15] wh ich appeared crimson red in colour however non-embryogenic cells appeared unstained, distorted and irregular in shape or with lighter shade due to lack of p rotoplasm. Squash preparation of the callus tissue of A. malaccensis showed that the cellular composition was heterogeneous ranging fro m small cells with dense cytoplasm to large cells with vacuolated cytoplasm in case of young or friable callus. The friab le callus cells were loose, with entire margin, full of protoplasm and mostly globular in shape with average diameter of 31.5µm. The other shapes like oval, slightly elongated, beaked or co mma shaped ranging between 28-45 µm in length and 18-35µm in breath were also observed ( Figure 2).
Oil extract ion fro m callus of agarwood had successfully been done earlier [12] and reported that the oil has about 30% similarities with that of agar oil ext racted fro m natural wood. In Taxus baccata L. Washingtonii, the valuable "Taxol", an anticancer agent is being ext racted fro m callus tissues [20]. As the A. malaccensis is valued for agar o il, the mass callus production shows great promises to extract essential oil d irectly in future without sacrificing the whole tree.

Conclusions
Fro m the present investigations, a reproducible, standard and efficient tissue culture protocol for callus production using leaf exp lants in A. malaccensis La m. has been defined which has importance fro m point of view of use of very less hormonal concentrations. MS mediu m supplemented with BAP (0.5 mg/l) + NAA (3mg/ l) at 4% sucrose concentration was found the best for obtaining maximu m callus bio mass in agar species. Overall, co mb ination A was found mo re suitable in terms of callus bio mass at 3-5% sucrose concentrations as compare to others. As the tree is economically important for preparation of perfu mes, incense and traditional medicines, the present investigations may be helpful for large scale production of callus biomass and to oil traders for synthesis of agar oil fro m it. Another interesting area of research may be to investigate effect of introduction of endophytic fungi in it and its effect on agar oil quality and quantity analysis.