In vitro propagation of Pterocarpus santalinus L. (Red Sandalwood) through tissue culture

Studies have been conducted to develop an in vitro propagation protocol for Pterocarpus santalinus L., an endangered medicinal plant. Stem cuttings excised from one year old plants were surface sterilized successfully using 15 % clorox (5.25 % NaOCl) for 10 minutes followed by 70 % ethanol for two minutes and established on Mc Cowns woody plant medium (WPM) with 0.1 % activated charcoal. Different explant types were tested including cotyledonary nodal segments, mesocotyl segments, in vitro derived shoot tips, immature and semi-hard wood cuttings detached from one year old plants and in vitro germinated seedlings. Maximum number of shoots and shoot branches were obtained with the Gamberg medium (B5) with 8.0 2.0 formed on micro shoots on half-strength solid Murashige and hours. In vitro rooted plantlets were successfully acclimatized in pots containing sand:coir dust (1: 1) in a humid chamber


INTRODUCTION
Red sandalwood, Pterocarpus santalinus L. (Fabaceae), is a highly valued woody plant with dark claret-red heartwood, which contains 16 % Santalin, a red colouring agent. Santal, pterocarpin and homopterocarpin are three other crystalline colouring agents present in the heartwood that are used in the pharmaceutical, paper, pulp, soap, food and textile industries to obtain a dark maroon to purple colour. In ayurvedic medicine, red sandalwood is used as an antiseptic, wound-healing agent, and in anti-acne treatments. Because of its hard texture, the red sandalwood is used in making furniture and in wood carvings. The natural habitat of P. santalinus is in the southern Ghats states in India, which is a hilly region with a hot dry climate. It is now considered as an endangered species and placed in the red list of endangered species under IUCN guidelines (International Union for Conservation of Nature, 2011) compelling the Indian government to ban exportation of this commodity. This decision has negatively affected Sri Lanka, as we have been importing red sandalwood from limited number of red sandalwood trees, which had been brought from India, are present, mainly in the southern region of the country (Arunakumara et al., 2005). Red sandalwood is conventionally propagated through seeds, coupled with low viability and seed dormancy due to the presence of phenolic compounds (Kumarasinghe et al., 2003).
The conventional vegetative propagation of red sandalwood has proven to be ineffective (Dayathilake et al., 2001) as the cuttings were found to be hard to root even with the application of growth regulators (Subasinghe et al., 2004). Therefore the conservation and largescale propagation of this endangered species has some inherent limitations. In this context tissue culture seems to be a promising technique for conservation and large scale multiplication of several woody species (Razdan, 1994). Studies on tissue culture of red sandalwood began in 1980s with more recent attempts to improve shoot sprouting using seedling explants in India (Anuradha for micropropagation of elite and quality trees of red sandalwood has not been reported to date in Sri Lanka.

March 2013
Journal of the National Science Foundation of Sri Lanka 41 (1) Therefore, an attempt was made to develop a feasible in vitro propagation protocol for red sandalwood through tissue culture techniques, to enable mass propagation of this plant for biodiversity conservation and to meet the increasing demand.

METHODS AND MATERIALS
Pods were picked directly from mature red sandalwood trees (over 25 years old) at the green to brownish stage. Seeds were surface sterilized using 10 % clorox for 20 min followed by 70 % ethanol for 2 min and cultured on Gamberg (B5) medium (Gamborg et al., 1968). Six different explant types were used for the study. In vitro germinated seedlings were excised after 20 d to derive mesocotyl segments, cotyledonatory nodal segments and shoot tips. Mother plants were maintained under plant-house conditions to excise stem cuttings. Fresh and immature terminal shoot cuttings and subsequent nodal shoot segments (semi-hard wood, 3-5 cm in length with dormant auxiliary buds) were collected during the early hours of the day at 2 wk intervals. In vitro germinated 20 day old seedlings were used as the control.
As a rejuvenation procedure, decapitation and spraying of 10.0 ppm 6-benzyle amino purine (BAP) solution was done for the mother plants, at 2 wk intervals. Plants were maintained under plant-house conditions free from pests and diseases and watered when necessary. Overhead watering was avoided. A 100.0 ppm solution of thiophanate methyl 70 % (topsin) was sprayed 24 h before collecting the buds for culturing to prevent fungal infections. Plants were treated with 200.0 ppm Albert's solution (complete fertilizer mixture) at 2 wk intervals.Cultures were kept aseptically in a growth room (23 ± 2 0 C temperature, 60 % RH) and cool photoperiod under 1220 lx of light intensity. All media contained 3.0 % sucrose and 0.01 % myo-inositol. The pH of the medium was adjusted to 5.8. Prepared media were autoclaved at 121 0 C and 1.05 kg cm -2 pressure for 20 min. Experiments were designed according to complete randomized design with 20 replicates in each treatment and all parametric data were analyzed using SAS statistical software. Mean separations were carried parametric data were analyzed using the Chi Square test.

Surface sterilization of shoots
Immature and semi-hard wood shoots excised from mother plants were immediately dipped in a beaker containing distilled water. In the laboratory, shoot segments were treated with liquid detergent (Teepol 5 % v/v) for 5 min and placed under running tap water for half an hour. Shoots were transferred to 0.3 % topsin solution and kept for 1 h, thoroughly washed twice using sterile distilled water and transferred to different concentrations of clorox separately (10 %, 15 % and 20 % v/v) for three different time durations (10,15 and 20 min). To enhance the activity of the detergent, two drops of Tween 20 (poly oxyethelene sorbitanmonolaurate) was mixed with each of the clorox solutions,introduced to 70 % ethanol for 2 min and washed twice using sterile distilled water. This procedure was repeated twice. Finally, cut ends of the explants were trimmed and implanted vertically on a basal culture medium [MS (Murashige & Skoog, 1962) or WPM (Lloyd & McCown, 1980)]. Percentages of non contamination, browning and survivals as well as the number of shoot buds initiated and the new leaves formed were recorded over a period of 4 wks.

Establishment of shoots
Surface sterilized immature and semi-hard wood shoots were cultured on MS (Murashige & Skoog, 1962) and WPM (Lloyd & McCown, 1980) media with and without 0.1 % activated charcoal. Percentages of browning and survivals as well as the number of shoot buds initiated and the new leaves formed were recorded over a period of 4 wks.

Effect of explant type, culture medium and combination of plant growth regulators on in vitro shoot proliferation
Separate experiments were conducted using six different types of explants. Explants were cultured on MS and B5 (Gamborg et al., 1968) media with different of shoots, mean shoot height, number of fully opened leaves and number of branches were measured at four week intervals up to the sixth sub-culture. media. Root initiation rate was measured after 4 wks by uprooting the plants.

Acclimatization of plantlets
In vitro rooted plantlets were transferred to pots containing sand, coir dust, cow dung and top soil in different ratios and placed under high humid conditions in the laboratory for 2 wks. Potted plantlets were transferred to planthouse and maintained under high humidity for another 4 wks (Figure 1a).
Plantlets were transferred to poly bags containing cow dung: top soil: sand (

Surface sterilization of shoots
The highest survival (80 %) was observed in immature cuttings treated with 15 % clorox for 10 minutes. Semihard wood cuttings gave the maximum survival rate of 40 % when treated with 10 % clorox for 20 minutes and 15 % clorox for 10 and 15 minutes (data not shown).

Establishment of shoots
There was a combined effect of media type, presence of activated charcoal and explant type on browning, which charcoal (0.1 %) incorporated WPM medium was the best culture initiation medium for immature and semihard wood cuttings where the browning of explants was reduced to zero and 10 %, respectively (Table 1). Note: Means within the last column followed by the same letter are not    (Table 2). However, the interaction between the type of the medium and the explant type leaves and shoot buds produced.

Effects of explant type, culture medium and combination of plant growth regulators on in vitro shoot proliferation
Interaction of culture medium type, BAP and NAA number of shoots produced by all explant types.
Interaction of medium type, BAP and NAA concentrations of cotyledonary nodal segments, mesocotyls, in vitro derived shoot tips, immature stem cuttings (Table 4) and the control, while semi-hard wood cuttings did not show any height increment during the four week duration of culture.
nodal segments, mesocotyls, in vitro derived shoot tips, immature and semi-hard wood cuttings) on B5 medium level (Table 5). However, in vitro germinated seedlings; number of branches (3.09 ± 0.22) on B5 medium with phase, sub-culturing was practiced at four week intervals up to the sixth sub-cultures. However, after the forth subculture, the number of new shoot production was reduced drastically (data not shown).

Effects of pulse treatment, IBA concentration and potting media on root formation of micro cuttings
Pulse treatment and IBA-containing solid medium roots produced and the mean length of the roots (Figures  3 & 4). The highest (4.10 ± 0.00) mean number of roots per shoot was produced when 2,500.0 ppm IBA solution was used for the pulse treatment with 12 hours exposure time, followed by subsequent culturing in half strength a b    IBA concentrations and exposure times used for the of the pulse treatment and the concentrations of the IBA-0.05) affected the browning of shoots as well (Table 6).
Pulse treatment with 25.0, 250.0 and 2,500.0 ppm IBA supplemented media, caused browning and subsequent deaths of shoots (Table 6).
When shoots were pulse treated with 2,500.0 ppm IBA for 12 hours followed by transferring to liquid half rooting was observed within four weeks of incubation. However, they were fragile in nature and did not show any further growth and turned brown ( Figure 6). Shoots pulse treated with 25.0 ppm and 250.0 ppm IBA for 12 hours followed by transferring to liquid half strength produced roots.
Shoots dipped in 1,000.0 ppm IBA solution for 12 hours and subsequently transferred into sand medium produced 40 % rooting while none of the other treatments produced roots (Figure 7).

Acclimatization of plantlets
An 80 % success rate was observed, when the plantlets were acclimatized in sand : coir dust (1:1) potting medium, with increasing plant heights and production of new leaves and shoot branches (data not shown). A two-stage acclimatization procedure (six weeks under high humidity and low light followed by another four weeks in a plant-house under shade) was needed prior to

Surface sterililzation procedure for shoots
During the present study, immature stem cuttings of P. santalinus were successfully surface sterilized using 15 % clorox with 10 minutes exposure time followed by 70 % ethanol for 2 minutes exposure time resulting 80 % non-contamination rate, 80 % survival rate and 100 % non-browning rate. The use of mercuric chloride for surface sterilization of explants has been reported in many research articles. Prakash et al. (2006) reported that double sterilization using 70 % ethanol for 2 minutes followed by 0. Note: Means within the last column followed by the same letter are not

Establishment of shoots
In the present study, survival rates (number of explants that remained green without contamination for four weeks after culture initiation) were found to be dependent on the type of the medium, presence of activated charcoal found to be the best medium with the highest survival rate when compared to MS medium. Incorporation of activated charcoal into the culture medium reduced the browning by absorbing phenolic compounds and other exudates, enhancing the growth performance of explants and showed higher survival rate when compared to the media without activated charcoal. Immature stem cuttings showed higher survival rate than semi-hard wood cuttings.
It has been demonstrated that leaching of phenolic compounds from cut ends of nodal explants into the liquid nutrient medium could be controlled by the combination of serial transfer technique at intervals of 24, 48, 72 and 96 hours and the incorporation of antioxidants (250.0 ppm ascorbic acid and 50.0 ppm citric acid) into the culture media for P. santalinus immature stem cuttings from 10 year old trees with 70 % survival (Prakash et al., 2006). In the present study, immature cuttings cultured on 0.1 % activated charcoal incorporated WPM medium achieved 100 % survival rate without browning and with higher numbers of new leaves and shoot buds, thus showing that the WPM medium incorporated with 0.1 % activated charcoal is a better establishment medium for immature stem cuttings of P. santalinus.

Effects of explant type, culture medium and combination of plant growth regulators on in vitro shoot proliferation
It has been shown that cotyledonary nodal segments achieved maximum shoot multiplication (7.83 ± 0.30) per explant within six weeks and that higher concentrations of BAP and Kinetin decreased the multiplication rates of Pterocarpus marsupium while incorporation of low along with BAP induced shoot bud initiation (Anis et al., 2005). Prakash et al (2006) reported that a combination of BAP and different auxins (IAA or NAA) did not improve the shoot proliferation of P. santalinus, whereas BAP and thidiazuron (TDZ) stimulated the production of medium for nodal explants. However, during the current study, cotyledonary nodal segments produced a higher mean number of shoots, (4. 95 ± 0.22 ) when compared to mesocotyls (4.00 ± 0.00), in vitro germinated seedlings; the control (3.25 ± 0.44), in vitro derived shoot tips (3.00 ± 0.00) and immature cuttings (2.95 ± 0.22) on (Table 3) within four weeks and up to the forth subculture while no multiplication was observed in semihard wood cuttings.
Anis et al (4.01 ± 0.06 cm) per shoot for P. marsupium using cotyledonary nodal segments and Prakash et al. (2006) TDZ produced shoots with 6-7 cm mean length per shoot for P. santalinus using nodal segments. During the TDZ gave a mean shoot height of 4.55 ± 0. 51 cm for mesocotyls, 4.00 ± 0.00 cm for immature cuttings, 3.92 ± 0.18 cm for in vitro excised shoot tips, 3.72 ± 0.25 for the control and 3.62 ± 0.22 for cotyledonary nodal segments (Table 4). These heights are capable of being transferred to further multiplication or root formation. Semi-hard wood cuttings did not show any height increment during the four week duration of culture.
Red sandalwood is a plant with a higher branching habit, producing side shoots frequently near the soil surface, not only due to apical bud damage but also when it bends due to shade conditions. This is common to in vitro cultures as well, with cotyledonary nodal segments producing more branches (4.20 ± 0.00) than mesocotyls (3.75 ± 0.71) ( Table 5) and controls (3.09 ± 0.22), and semi-hard wood cuttings producing more branches (3.45 ± 0.51) (Figure 2b) than in vitro derived shoot tips (2.50 ± 0.51 and immature cuttings (3.15 ± 0.58) ( Table 5), which may be governed by the phenomenon of apical dominance. To obtain the above results, explants have to cuttings to form roots. B5 me NAA was the best medium and plant growth regulator combination to form the highest number of new shoots and new shoot branches from cotyledonary nodal segments, mesocotyls, in vitro derived shoot tips, immature and semi-hard wood cuttings and in vitro germinated seedlings, respectively within a four week interval. Mesocotyls gave the highest mean shoot height under the same conditions. The regeneration ability was reduced drastically after the forth sub-culture.

Effects of pulse treatment, IBA concentration and potting media on root formation of micro cuttings
Stem cuttings even treated with higher concentrations of auxins up to 3000.0 ppm failed to produce roots (Subasinghe et al., 2004). Sanjaya et al (2006) reported that the best results for S. album shoots were given when hours to induce rooting and subsequently cultured on MS medium. Isolated micro shoots of P. marsupium have been rooted using a strategy of giving pulse treatment of Prakash et al. (2006) has reported that P. santalinus shoots derived from in vitro multiplication were rooted IBA, failed to produce more than one root. During the current study, it was seen that without pulse treatment, IBA) concentrations were not effective in root formation. Stem cuttings with 2.0 cm length treated with 250.0 ppm IBA solution for 12 hours followed by transferring to formed roots successfully.

Acclimatization of plantlets
The present study revealed that in vitro rooted plantlets could be successfully acclimatized in potting media containing sand : coir dust 1:1 ratio. Eighty percent survival was observed when the plantlets were placed maintained under plant-house conditions for another four Somewhat similar results were recorded by Prakash et al., (2006) where 70 % survival was seen among in vitro rooted plantlets of P. santalinus acclimatized in a plant-growth chamber at 25 ± 2 0 C and 85 % RH under tubes for two weeks. The plantlets were then transferred to a shady area of a forest nursery for another four weeks after repotting in larger 20 cm pots containing a mixture of soil and farmyard manure (4:1). It is therefore seen that the acclimatization procedure developed in the current study could be effectively applied to acclimatize in vitro rooted plantlets of P. santalinus. It can be concluded that Pterocarpus santalinus could be propagated on massscale using the micropropagation protocol reported in demand for this plant