Red sandalwood (Pterocarpus santalinus L. f.): biology, importance, propagation and micropropagation

Pterocarpus santalinus L. f. (Fabaceae; red sanders) is prized for its wood whose colour and fragrance is due to the presence of santalins that have pharmaceutical and industrial uses. Red sanders is listed as an endangered plant species on the IUCN red data list as a result of the exploitation of its wood and essential oil. This review emphasizes the pollination biology, seed germination, vegetative propagation and micropropagation of P. santalinus. Excessive use of P. santalinus has also caused the emergence of various adulterants, so accurate identification is essential.

, and as a textile dye (Gulrajani et al. 2002). The medicinal properties of P. santalinus have been extensively reviewed elsewhere (Navada and Vittal 2014;Azamthulla et al. 2015) and will not be covered in this review. However, multiple uses (Table S2), ethnomedicinal uses (Table S3), and phytochemistry (Table S4) have been provided as supplementary tables to offer a more rounded appreciation of this tree in the context of this review.
The texture and colour differentiate good quality from poor quality trees, with ''wavy grain wood texture with intense red color'' in the former and ''straight grain wood texture with light red color'' in the latter (Prakash et al. 2006), and it is the superior quality of P. santalinus that makes it popular in the furniture industry (Prakash et al. 2006;Arunkumar et al. 2011;Arunkumar and Joshi 2014;Azamthulla et al. 2015). In Japan, P. santalinus is used to make carvings and musical instruments, shamisen and koto (Kukrety et al. 2013b;Arunkumar and Joshi 2014;Azamthulla et al. 2015;Ramabrahmam and Sujatha 2016), as well as name seals or hankos. In Buddhism, P. santalinus is considered to be a symbol of holiness, and is thus used for carved statues, as a constituent of incense (Wu et al. 2011), and for cremation (Ramakrishna 1962). In China, P. santalinus wood has a long history of use in furniture and other valuable wood products (Berliner 1996;Kaner et al. 2013).
The export of P. santalinus from India to Europe started in the 17th century, mainly for fabric dyeing (Vedavathy 2004). The Herbal Folklore Research Centre in Tirupati, India, estimated that from 500 planted trees ha -1 , at least 500 kg of heartwood per tree can be obtained after 25 years, thus 25 t ha -1 of wood plantation (Vedavathy 2004). At 2004 prices of Rs. 75 kg -1 , such a plantation would yield a return of Rs. 177.5 lakhs ha -1 (US$375,000 ha -1 ) (Vedavathy 2004). Current market prices are, however, unknown to the authors, although prices are likely to be high since natural P. santalinus stands have been in decline as a result of this overexploitation for commercial purposes, earning it an endangered status since 1997 (IUCN 2018).
This review provides an overview of the reproductive biology, seed germination and micropropagation of P. santalinus as tools for its conservation and large-scale propagation.

Basic flowering biology, and sexual and vegetative reproduction Pollination and seedset
The P. santalinus tree flowers in the dry season (Rao et al. 2001;Rao and Raju 2002). The flowers are papilionaceous, bisexual, large and and yellow (Rao and Raju 2002).
Flowering is discontinuous, blooming at intervals of 2-5 d (Rao et al. 2001). Flowers open at night and the primary pollinators are Apis dorsata, A. cerana indica and A. florea (Rao and Raju 2002). P. santalinus, which shows facultative xenogamy, tends to eliminate growing fruits from self-pollinated flowers, i.e., there is large-scale abortion of flower buds, flowers and fruit (Rao et al. 2001), and has very low fruit set (-6%), 52% of which set seed (Rao and Raju 2002).

Seed germination
Traditional seed propagation of P. santalinus yields low germination percentages due to a hard testa, poor viability, and sensitivity to temperature (Kumar and Gopal 1975;Dayanand and Lohidas 1988;Anuradha and Pullaiah 1998;Naidu 2001a, b;Naidu and Rajendrudu 2001). Dried, soaked and scarified P. santalinus pods resulted in 49% germination (Kumarasinghe et al. 2003) although seed germination in natural stands or under artificial propagation is generally low (-30%) (Kumar and Gopal 1975;Dayanand and Lohidas 1988;Kalimuthu and Lakshmanan 1995;Naidu 2001a, b;Naidu and Rajendrudu 2001). Alternate wetting and drying every 48 h enhanced germination, reaching 73% (Vijayalakshmi and Renganayaki 2017). Seed germination, seedling height, and root collar diameter were all significantly stimulated by fire (Kukrety et al. 2013b). Presoaking P. santalinus pods with 500 mg/L gibberellic acid for 24 h resulted in 66.7% seed germination, as well as improved plant growth and seedling survival relative to other treatments with tap water, luke warm water, gibberellic acid, H 2 SO 4 or HCl (Patel et al. 2018).

Vegetative propagation
Vegetative propagation of P. santalinus by semi-hardwood cuttings, cleft grafting, or air layering is not able to produce stock numbers required for effective preservation or for commercial purposes (Kedharnath et al. 1976;Kesava Reddy and Srivasuki 1990). Relative to seed germination, there are almost no studies on ex vitro vegetative propagation for red sanders. However, to provide elite germplasm with desired traits, such as the wavy grain or phytochemicals such as santalins, vegetative propagation under controlled conditions is desirable, and in vitro propagation allows for the production of true-to-type plants via micropropagation such as axillary shoot multiplication or shoot tip culture at a large scale, to continuously produce plantlets with uniform characteristics. In tree biotechnology, such as for Indian sandalwood (Teixeira da Silva et al. 2016b), in vitro propagation also allows for the improvement of desired characteristics such as pathogen resistance or improved wood quality by genetic engineering. The next section assesses the progress of micropropagation of P. santalinus.

Micropropagation Explants
The explant source (i.e., mother plant) and the procedure to surface disinfect explants are important aspects underlying the success of a tissue culture protocol (Leifert et al. 1994;Teixeira da Silva et al. 2016c). Information about the explants used for the in vitro propagation of P. santalinus, as well as surface disinfection protocols, are summarised in Table 1. P. santalinus seedlings derived from in vitro seed germination have been a popular source of explants, while in vitro germinated seedlings, shoot tips, cotyledons, hypocotyls, mesocotyl and nodes have also served as popular sources of explants for culture initiation since they do not require surface disinfection (Lakshmi Sita et al. 1992;Anuradha and Pullaiah 1999a,b;Chaturani et al. 2005;Rajeswari and Paliwal 2008;Balaraju et al. 2011;Vipranarayana et al. 2012;Warakagoda and Subasinghe 2013). In terms of ex vitro sources of explants, Prakash et al. (2006) used young terminal shoot cuttings collected from mature trees in winter as the explant; Ashrafee et al. (2014) used leaf segments from 1 to 2 year old plants while Sarita et al. (1988) used nodes and terminal cuttings.

Basal medium
The most commonly used and preferred basal medium for in vitro studies on P. santalinus is Murashige and Skoog (1962) (MS) medium (Table 2). Lakshmi Sita et al. (1992) used Gamborg's B 5 medium (Gamborg et al. 1968) with 2% sucrose and 0.8% agar to multiply axillary shoots from shoot tips derived from seedlings germinated in vitro. Chaturani et al. (2006) used Anderson medium (Anderson 1980) and Vitis medium (Chee and Pool 1987) to germinate seed. Anuradha and Pullaiah (1999a) employed halfstrength B 5 medium supplemented with 0.05% activated charcoal to germinate seeds in vitro then used B 5 medium with 8.88 lM 6-benzyladenine (BA) for shoot tip culture.

In vitro propagation from predetermined meristems
Three predetermined meristems were employed in P. santalinus tissue culture: shoot tips, cotyledonary nodes and nodes from mature trees (Table 2). Shoot tips from in vitro germinated seedlings were used by Lakshmi Sita et al. (1992), Anuradha and Pullaiah (1999a), and Balaraju et al. (2011) for shoot tip culture, with either BA as the most effective cytokinin (Lakshmi Sita et al. 1992; Anuradha and Pullaiah 1999a) or a combination of BA and thidiazuron (Balaraju et al. 2011). Cotyldenory nodes were sucessfully applied for the in vitro propagation of P.
santalinus (Anuradha and Pullaiah 1999a;Arockiasamy et al. 2000;Rajeswari and Paliwal 2008;Warakagoda and Subasinghe 2013). BA, alone or combined with other cytokinins or auxins, has frequently been utilized for the micropropagation of P. santalinus (Table 2). Prakash et al. (2006) cultured nodes of mature trees directly on filter paper bridges employing liquid MS medium containing 1.16-9.30 lM kinetin or 1.11-8.88 lM BA, as well as an antioxidant, observing that 4.44 lM BA was optimum for bud break and shoot multiplication.
In vitro propagation (callogenesis, regeneration and somatic embryogenesis) Leaf, cotyledon, root, internode and nodal segments (presumably with axillary buds) from in vitro P. santalinus seedlings formed callus, but shoot regeneration was not reported (Chaturani et al. 2005). Callus was induced from leaves and internodes of P. santalinus by Ashrafee et al. (2014) solely to assess antibiotic activity against Aeromonas and Pseudomonas but regeneration was not assessed. Details of effective plant growth regulator concentrations and combinations, medium composition, and explant type, as well as their effects on morphogenesis are presented in Table 2.

Rooting and acclimatization
Sucessful roooting of in vitro-raised plants followed by effective acclimatization and successful transfer of in vitropropagated plants to field conditions is the final objective of any micropopagation protocol and care is needed to avoid hyperhydricity in in vitro-raised plants, which tend to display poor rooting efficiency (Ruffoni and Savona 2013;Teixeira da Silva et al. 2017b). Rooting and acclimatization protocols for in vitro-raised shoots of P. santalinus are summarized in Table 2. Only a few studies have quantified the survival of micropropagated plants (Lakshmi Sita et al. 1992;Prakash et al. 2006;Rajeswari and Paliwal 2008;Balaraju et al. 2011;Warakagoda and Subasinghe 2013). Among the 12 reports on P. santalinus tissue culture, in vitro rooting employed full-strength, half-strength and quarter-strength MS medium (Table 2). According to Arockiasamy et al. (2000), quarter-strength MS medium supplemented with 5.71 lM IAA was effective for 76.2% rooting of shoots derived from cotyledenory nodes, but acclimatization and survival of plantlets were not reported. Vipranarayana et al. (2012) applied a pulse treatment of 7.34 lM IBA in half-strength MS but details about survival were not reported. Rajeswari and Paliwal (2008) achieved 85% plantlet survival ex vitro after a pulse treatment of 5 lM IAA and 1 lM IBA for 25 d. In contrast, Warakagoda and Subasinghe (2013) showed limited success (46%

Variability in quality and quality control
There is a problem with the adulteration and falsification of plant material in the P. santalinus market. The heartwood of Adenanthera pavonina Willd. (Mimosaceae), known as 'Ranjana' and 'Raktakambal' in West Bengal and 'Bari Gumchi' in the northern parts of India, is often sold as a fake substitute for P. santalinus, while artificially colored wood shavings and the sawdust of some other trees are also sold on the market as cheap substitutes (Botanical Survey of India 2012). In China, the manufacture of furniture utilizes Dalbergia louvelii R. Vig. (violet rosewood) as a substitute for P. santalinus since both plants have a very similar appearance and anatomical characteristics, and cheaper D. louvelii is often illegally used to impersonate the valuable P. santalinus (Zhang et al. 2014). Zhang et al. (2014) used conventional infrared spectroscopy (FT-IR), second derivative infrared (SD-IR) spectroscopy and twodimensional correlation infrared (2DCOS-IR) spectroscopy to differentiate furniture made of P. santalinus wood from furniture made from D. louvelii. They observed that P. santalinus wood had a higher holocellulose content than D. louvelii wood while D. louvelii had more NaOH-and benzyl-alcohol-based extracts than P. santalinus. The size and age of trees affects the heartwood content and wood density of P. santalinus (Suresh et al. 2017).
Woody anatomy such as grain waviness can be used to delimit and identify P. santalinus (Rawat and Uniyal 1996;Gasson and MacLachlan 2010). The Botanical Survey of India (2012) used various anatomical methods such as maceration, scanning electron microscopy, exo-and endomorphic features, and fluorescence analysis to correctly identify P. santalinus wood samples.
Molecular markers are regularly utilized to measure the degree of genetic variation within natural or breeding populations, and have been extensively used in Indian sandalwood research (Teixeira da Silva et al. 2017a). In P. santalinus, RAPD (random amplified polymorphic DNA)based marker analysis was used to detect variations in micropropagated plants raised from shoot tips, verifying that in fact no variation existed (Balaraju et al. 2011). RAPD was also used by Usha et al. (2013) to detect variation among nursery-grown plants. Variation in genetic distance among natural accessions, detected by RAPD markers, reflected a high level of DNA polymorphism due to outcrossing (Padmalatha and Prasad 2007;Usha et al. 2013). Jhansi Rani and Usha (2013) developed a sequence characterized amplified region (SCAR) marker to differentiate wavy from straight-grained plants at the seedling stage. Jyothi et al. (2014) reported differences in the quantity of genomic DNA in samples collected from different locations in Andhra Pradesh, India.
Therefore, quality control, as assessed by anatomical or chemical methods, is essential to verify the originality of P. santalinus wood while molecular methods serve to confirm genetic stability.  coir dust (1:1) was 80% when kept in humid conditions and weak light for the first 4 w then for 6 w in greenhouse Warakagoda and Subasinghe (2013) Red sandalwood (Pterocarpus santalinus L. f.): biology, importance, propagation and… 751

Conclusions and future perspectives
This review highlights key advances in the tissue culturebased biotechnology of economically important Pterocarpus santalinus. To date, effective protocols for seed surface disinfection and in vitro germination exist. There are also effective protocols for direct shoot regeneration from a range of explants or through callus induction. In most cases, explants are derived from seeds or seedlings which are not suitable for clonal propagation (Table 1). Therefore, a clonal method should be developed from vegetative tissues of elite germplasm. Rooting and survival of micropropagated plants remain a major limitation to the success of P. santalinus tissue culture and should be optimized in the future, for example by using CO 2 enrichment and vessels that allow for maximized aeration without impacting relative humidity levels within the culture vessel (Teixeira da Silva et al. 2005). The ability to stably produce units that allow for germplasm conservation would then stimulate the need for cryoconservation (Teixeira da Silva and Engelmann 2017; Bi et al. 2017), including through the application of synthetic seeds (Sharma et al. 2013). Analytic hierarchy, which is a multicriteria decision-making tool, is valuable for incorporating the perceptions of stakeholders when planning the conservation and restoration of a P. santalinus population (Kukrety et al. 2013a, c). The micropropagation and biotechnology of another commercially important tree in this genus, P. marsupium (Indian kino tree), have recently been reviewed (Teixeira da Silva et al. 2018).