The geometric parameters of the title compound, C4H3BrN2O, are in the usual ranges. The crystal packing is characterized by N—H⋯N and C—H⋯O hydrogen bonds and short O⋯Br contacts

Ashalatha, B. V.

Bolte, Michael

Narayana, B.

Sarojini, B. K.

Yathirajan, H. S.

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ORIGINAL ARTICLEJ For Res (2006) 11:147–151 © The Japanese Forest Society and Springer-Verlag Tokyo 2006DOI 10.1007/s10310-005-0208-1Sanjaya · Bagyalakshmi Muthan · Thrilok Singh RathoreVittal Ravishankar RaiFactors influencing in vivo and in vitro micrografting of sandalwood(Santalum album L.): an endangered tree speciesReceived: September 28, 2004 / Accepted: May 12, 2005Abstract In vivo micrografting of Santalum album wasachieved (50%) by grafting 4- to 5-cm-long scions, collectedfrom a candidate plus tree (CPT) of 50–60 years of age, onto90-day-old nursery-grown rootstock. Scion size, rootstockage, and scion collection season were found to influencegraft success. Grafted plants were incubated under green-house conditions for 6–8 weeks during the graft union pro-cess. In vitro micrografts were achieved by placing 1- to2-cm-long scions derived from nodal shoot segments (col-lected from CPT) onto the hypocotyl of 45-day-old in vitrorootstocks. Use of in vitro grown shoots as a source of sciongave better graft success (60%) than scions collected di-rectly from field-grown trees. In vitro grafting was also influ-enced by scion size and rootstock age. Under favorableconditions, scions and hypocotyls unite to form completeplants that produced two to four leaves after 6–8 weeks.This is the first report on in vivo and in vitro micrograftingof S. album having potential for production of disease-freeclonal plants for conservation and improvement targets.Key words Graft success · Mature tree · Micrografting ·Scion · RootstockIntroductionSantalum album, L. family Santalaceae, is commonly knownas Indian sandalwood or Chandan. It is a small, evergreenhemiroot parasite tree, which attains a height of 12–13mSanjaya · T.S. RathoreTree Improvement and Propagation Division, Institute of WoodScience and Technology, Malleshwaram, Bangalore, IndiaSanjaya (*) · B. MuthanInstitute of Bio-Agricultural Science, Academia Sinica, Nankang,Academia Road, Taipei 11529, Taiwan, ROCTel. +886-22-789-8626; Fax +886-22-651-1164e-mail: sanjayadagoor@yahoo.co.inV.R. RaiDepartment of Studies in Applied Botany and Biotechnology,Manasagangothri, University of Mysore, Mysore, Indiaand a girth of 1.0–2.4m. Sandalwood is highly valued for itsfragrant heartwood and sandal oil used in world-class per-fumes, cosmetics, and medicine (Ananthapadmanabha2000). Sandalwood is also highly prized next to ivory forcarving (Srinivasan et al. 1992).Natural regeneration of sandalwood occurs mainly byseeds and also through root suckers. However, seedlings areextremely heterozygous due to outbreeding (Srimathi et al.1995). Vegetative propagation through root suckers orcuttings has been reported by Uniyal et al. (1985). In vitroregeneration has been reported through somatic embryo-genesis using explants from seedlings as well as from field-grown trees (Lakshmi Sita 1986; Rao and Bapat 1995;Rai and McComb 2002). However, S. album is stronglyrecalcitrant to in vivo and in vitro propagation forwhich only limited success has been achieved to date(Uniyal et al. 1985; Rao and Bapat 1992). In vitro rootinglimits widespread application of micropropagationtechniques for tree improvement of sandal. Vegetativepropagation of plus trees for the establishment of clonalseed orchards is achieved by cleft grafting on 1- to 2-year-old seedlings (Srinivasan et al. 1992). This method, how-ever, is limited by low graft success and plagiotropic growthof developing scions. To date and as far as we are aware,vegetative propagation from branch cuttings and in vitroclonal propagation of superior genotypes have not beenreported. Overexploitation, sandal spike disease (caused bya mycoplasma-like organism), failure of regeneration ef-forts, and illicit felling have narrowed the gene pool of thisheritage species that is now more and more threatened in itsnatural habitat. There is therefore a need to develop alter-native clonal techniques to produce disease-resistant andhigh oil-yielding clones for urgent conservation and localuses.The process and application of micrografting for over-coming incompatibility problems was demonstrated byJonard (1986). This technique has been also used forrejuvenation and production of clonal plants in Sequoiasempervirens (Huang et al. 1992), Acacia mangium(Monteuuis 1995), Morus alba (Fengtong et al. 1996), andTheobroma cacao (Mallika et al. 1997). The objective of the148present study was to assess the influence of different factors,i.e., scion size, rootstock age, and season to produce se-lected sandalwood clones by the use of in vivo and in vitromicrografting techniques.Materials and methodsPreparation of rootstocksFresh seeds were collected from Nalla clonal seed orchardin Bangalore, India. Decoated seeds were treated with gib-berellic acid (GA3) (0.05% w/v) solution overnight andsown to produce seedlings that were raised in 250-ml or450-ml single-cell root trainers filled with potting medium(compost 50%, sand 40%, and garden soil 10%) and main-tained in a greenhouse (25° ± 5°C). Twenty replicates foreach different age group of rootstocks (40- to 110-day-old-seedlings) were used for in vivo micrografting.MS basal medium (Murashige and Skoog 1962) supple-mented with 3% sucrose and 0.6% agar was used formicrografting. The pH of the medium was adjusted to 6.0with 0.1 N NaOH solution before autoclaving. The medium(25ml) was dispensed into 25 culture tubes (150mm;Borosil, India). The tubes with media were autoclaved at 15psi and 121°C for 20min. Seeds treated overnight with GA3were surface-sterilized with 0.075% (w/v) mercuric chloridefor 6–7min followed by washing with sterile distilled water(6–8 times) under aseptic conditions before inoculation(one per tube) onto the autoclaved medium. Cultures wereincubated at 28° ± 1°C temperature and under a 12-hphotopheriod of 60mmolm−2 s−1 light intensity provided bycool-white fluorescent tubes (Philips, India). Twenty repli-cates for each different age group (20–60 days old) wereused as rootstocks for in vitro grafting.Preparation of scionsActively growing shoots with apices were collected from ahigh oil-yielding (4%–5%) 50- to 60-year-old candidate plustree (CPT) growing on the campus of the Institute of WoodScience and Technology, Bangalore, India, and were dippedin 0.1% (w/v) fungicide (Bavestin) for 3–4min and thenwashed with distilled water. These shoots were used to de-termine suitable scion size for in vivo micrografting.Nodal shoot segments (2.5–3.5cm in length) with dor-mant axillary buds were collected from the same CPT, sur-face sterilized with 70% (v/v) ethanol for 30–40s, followedby 0.075% (w/v) mercuric chloride (Hi-media, India) for 5–6min. Surface-sterilized explants were washed (6–8 times)with sterile distilled water and inoculated onto MS mediumsupplemented with a-naphthalene acetic acid (NAA)(0.53mM) and benzylamino purine (BA) (11.09mM) formultiple shoot induction (MSI). All the cultures were incu-bated at 28° ± 1°C under a 12-h photoperiod with a lightintensity of 60mmolm−2 s−1 provided by cool white fluores-cent lamps. Subculturing was carried out at intervals of 4weeks onto fresh MSI medium for production of shoots. Invitro shoots derived from third subculture of 0.5 to 2.0cmin length (scion) were used for the various in vitromicrografting experiments (20 replicates each). In anotherset of experiments, 20 shoot tips of 1.0–2.0cm in length thatwere collected directly from a CPT and were dipped in0.1% (w/v) fungicide (Bavestin) for 3–4min followed by0.075% (w/v) mercuric chloride (Hi-media) for 5–6min.Surface-sterilized explants were washed (6–8 times) withsterile distilled water and were also used as scion for in vitromicrografting.In vivo micrograftingAn incision of 0.5–1.0cm was made on the decapitatedgreenhouse-grown seedling (40–110 days old) using a surgi-cal blade. Shoot apices derived from a plus tree were in-serted into the incision, which was then held together withthe help of an elastic strip. Scions (shoots) collected fromthe same CPT during winter (November–January) and themonsoon period (May–July) were used to study seasonaleffects on in vivo micrografting.In vitro micrograftingUnder aseptic conditions, a vertical split measuring0.25–1.0cm in length was made on the top of the de-capitated rootstock (20–60 days old) using a surgical blade.In vitro grown scions derived from nodal shoot segments(0.5–2.0cm long) and shoot apices (collected directly froma CPT) were inserted into the rootstock incision.After grafting, plants were transferred to culture tubescontaining liquid half-strength MS medium with 2%sucrose. The radicle of each grafted plant was pushedthrough the hole in a filter paper bridge to support theyoung plant.Hardening and transplantingIn vivo grafted plants were covered with polythene bags tominimize moisture loss and were maintained at 25° ± 5°Cunder greenhouse conditions. The percentage of survivaland growth of scions were determined after 8 weeks.In vitro micrografted plants were covered with polythenebags, incubated at 25° ± 1°C with continuous light(60mmolm−2 s−1) provided by cool white fluorescent tubes,and observed periodically to evaluate scion and rootstocksurvival. Well-established in vivo grafted plants with ex-panded leaves were transferred to a shade house and subse-quently moved to a nursery. After 8 weeks, in vitro graftedplants with two to four expanded leaves from the scion weretransplanted into 450-ml single-cell root trainers containingsterilized soilrite (Chougule, Bangalore), maintained at 25°± 1°C under continuous light (60mmolm−2 s−1) provided bycool white fluorescent tubes. Established plants were trans-ferred into the greenhouse, and then 2 weeks later weremoved to the nursery. Overall procedures of micrograftingare shown in Fig. 1.149Data analysisObservations were recorded for 20 grafted plants in eachtreatment. The survival percentage was determined bycounting the number of grafted plants surviving out of thetotal number of grafted plants at the end of the eighth week.ResultsIn vivo micrograftingThe graft success of in vivo micrografting varied from 20%to 50%. As depicted in Table 1, the length of the graftedscion had a significant effect on graft success. Graft successwas higher (50%) when the scion size was large (4.0–5.0cm)and low when the size was small (0.5–3.0cm). The effect ofdifferent age group rootstocks was examined (40- to 110-day-old seedlings); higher graft success (50%) was observedwhen 90-day-old rootstocks were used for grafting (Fig. 2).The percentage of graft success was almost uniform (40%)when 70-, 80-, 100-, and 110-day-old rootstocks were used(Table 2). On comparing success of grafts carried out duringdifferent seasons using 4.0- to 5.0-cm-long scions and 90-day-old rootstocks, the grafts made during winter estab-lished relatively faster and with high success rate. Low graft         GA3 treated                                      0.5–5 cm long In vivo          mature                                             shoot apices from                            germinated          seeds                                               mature tree                                       seedling Surface In vitro germi-nated seedlingson MS mediumstock)Scion In vitro multipleshoot production (4–5 cm in length)          In vivo          micrografting Scion(1–2 cm in length)        In vitro micrograftingIn vivo Micro grafted plant After 6–8weeks After 6–8weeks  In vitro graftedplants in greenhousesterilizationShoot apicesIn vivo grafted plants in greenhouseIn vitro micrografted plantsIn vitro decapi-tated seedling(root stock)(45-days-old)Decapitatedseedling (root stock)(90-days-old)Fig. 1. A schematic representation of in vivo and in vitro micrograftingin Santalum album. GA3, gibberellic acidA         BC DE FFig. 2A–F. In vivo micrografting of S. album. A Incision of root stock.B Positioning of the scion. C, D Applying elastic strip to protect thegrafted zone. E Large number of grafted plants in greenhouse. F Com-pletely established grafted plant (arrow indicates grafted zone)Table 2. Effect of age of rootstock on the success of in vivomicrografting in S. albumRoot stock age Number of grafted Graft success (%)(days) plants recovered40 4 2045 4 2050 6 3060 6 3070 8 4080 8 4090 10 50100 8 40110 8 40Scion size 4–5 cm with 4–6 leaves. Number of rootstocks grafted: 20.Data recorded at the end of eighth weekTable 1. Effect of scion size on the success of in vivo micrografting inSantalum albumShoot/scion length Number of grafted Graft success (%)(cm) plants recovered0.5–1.0 0 01.0–2.0 4 202.0–3.0 4 203.0–4.0 8 404.0–5.0 10 50Rootstock age 90 days. Number of rootstocks grafted: 20. Data re-corded at the end of eighth week150success was observed when the scions were grafted duringthe monsoon season (Fig. 3).In vitro micrograftingTable 3 shows the effect of scion size on in vitromicrografting; higher graft success (60%) was observed forscions of 1.5–2.0cm in length grafted on 45-day-oldrootstocks (Fig. 4). Low graft success was observed whenthe scion length was 0.4–0.5cm, and 1 week after graftingmost of the scions had turned brown. The age of rootstockalso influenced graft success; 60% graft success was re-corded when the 45-day-old rootstock was grafted with sci-ons of 1.5–2.0cm in length (Table 4). No difference in graftsuccess (50%) was observed when 35-, 40-, 50-, and 60-day-old rootstocks were used.DiscussionUp to a certain stage, maturation of trees is usually accom-panied by a decline of vegetative vigor. Adult trees requirerejuvenation treatments for recovering physiological statusmore favorable to rooting and true-to-type propagation.Rejuvenation can be achieved by micrografting shoot api-ces (microscions) from selected adult trees onto very youngrooted juvenile rootstock in vitro or in vivo (Emmarold andMantell 2001). Higher graft success and growth was ob-served for in vitro grafted plants when compared with invivo grafted plants, which might be due to juvenility of invitro derived scions. Low success of in vivo micrograftingmay be due to rootstock and scion age, injury to scionduring grafting, and water loss, especially in the smallestscions (0.5–1.0cm long). Graft success rate was compara-tively increased with scion length and no graft union wasobserved with the smallest scion (0.5–1.0cm). Similar obser-vations were reported for Gossypium hirsutum (Jinhua andGould 1999).The success of in vivo grafting declined with increasedage of rootstocks (90 days old and onward). This may bedue to active cytodifferentiation and lignification in the0102030405060Nov-Jan Feb-April May-july Aug-Oct% of graft successFig. 3. Effect of season on in vivo micrografting in S. album. Scionsof 4.0–5.0 cm in length were grafted on 90-day-old rootstocks. Datarecorded at the end of the eighth week with 20 replicates for eachtreatmentA              BC            DFig. 4A–D. In vitro micrografting of S. album. A, B Development ofscion on root stock. C Close view of point of attachment. D Completelyestablished grafted plantTable 4. Effect of rootstock age on the success of in vitro micrograftingin S. albumRoot stock age (days) Number of grafted Graft success (%)plants recovered20 4 2030 4 2035 10 5040 10 5045 12 6050 10 5060 10 50Scion size 1.5–2.0 cm long. Number of rootstocks grafted: 20. Datarecorded at the end of eighth weekTable 3. Effect of scion size on success of in vitro micrografting in S.albumShoot/scion Number of grafted Graft success (%)length (cm) plants recovered0.4–0.5 2 100.5–1.0 8 401.0–1.5 9 451.5–2.0 12 60Rootstock age 45 days. Number of rootstocks grafted: 20. Data re-corded at the end of eighth week151hypocotyl region and also due to less wound-healing effect.This indicates that sandal is quite weak in scion capacity toproduce callus, which helps ultimately in graft success. InCamellia sinensis (Prakash et al. 1999), maximum graft suc-cess was achieved in 4-month-old rootstock and low graftsuccess was recorded when 12-month-old rootstocks wereused. The season also affected the success of in vivo graft-ing, which was evident by our observations. Winter andearly summer (spring) favored graft union with fewer occur-rences of scion rotting. This method provides a cost-effective technique for clonal propagation and could beused in graft-based phase change and long-distance signal-ing studies in sandalwood and other tree species.Acknowledgments This study was supported by the World BankFREE (Forestry Research Education and Extension) Project. Theauthors are grateful to Prof. G. Lakshmi Sita, Department of Microbi-ology and Cell Biology, Indian Institute of Science, Bangalore, India,for critical comments and valuable suggestions during preparation ofthe manuscript.Literature citedAnanthapadmanabha HS (2000) Sandalwood and its marketing trend.My Forest 36:147–152Emmarold EM, Mantell SH (2001) In vitro micrografting of cashew.Plant Cell Tiss Org 66:49–58Fengtong M, Guo C, Liu Y, Li M, Ma T, Mei L (1996) In vitro shoot-apex grafting of mulberry (Morus alba L.). Hortscience 31:460–462Huang L-C, Lius S, Huang B-L, Murashige T, Mahdi EM, Van GundyR (1992) Rejuvenation of Sequoia sempervirens by repeated graftingof shoot tips onto juvenile rootstocks in vitro. Plant Physiol 98:166–173Jinhua L, Gould JH (1999) In vitro shoot-tip grafting improves recov-ery of cotton plants from culture. Plant Cell Tiss Org 57:211–213Jonard R (1986) Micrografting and its applications to tree improve-ment. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry:trees vol 1. Springer, Berlin Heidelberg New York, pp 31–48Lakshmi Sita G (1986) Sandalwood (Santalum album L.). In: BajajYPS (ed) Biotechnology in agriculture and forestry: trees vol 1.Springer, Berlin Heidelberg New York, pp 363–374Mallika VK, Joseph J, Chacko D, Vijaykumar NR, Nair VR (1997)Micrografting-a simple technique for quick field transfer of tissueculture derived plants in Cocoa (Theobroma cacao L.). In:Ravishankar GA, Venkataraman LV (eds) Biotechnological appli-cations of plant tissue and cell culture. Oxford IBH, New Delhi, pp101–107Monteuuis O (1995) In vivo grafting and in vitro micrografting ofAcacia mangium: Impact of ortet age. Silvae Genet 44:190–193Murashige T, Skoog F (1962) A revised medium for rapid growth andbioassay with tobacco tissue cultures. Physiol Plant 15:473–497Prakash O, Sood A, Sharma M, Ahuja PS (1999) Graftingmicropropagated tea (Camellia sinensis O. Kuntzel) shoots on teaseedlings – a new approach to tea propagation. Plant Cell Rep18:883–888Rai RV, McComb J (2002) Direct somatic embryogenesis from matureembryos of sandalwood. Plant Cell Tiss Org 69:65–70Rao PS, Bapat VA (1992) Micropropagation of Sandalwood (Santalumalbum L.). In: Bajaj YPS (ed) Biotechnology in agriculture andforestry. High-tech and micropropagation II, vol 18. Springer, BerlinHeidelberg New York, pp 193–210Rao PS, Bapat VA (1995) Somatic embryogenesis in Sandalwood(Santalum album L.). In: Jain S, Gupta P, Newton R (eds) Somaticembryogenesis in woody plants, vol 2. Kluwer, Netherlands, pp 153–170Srimathi RA, Venkateshan KR, Kulkarni HD (1995) Guidelines forselection and establishment of seed stands, seed production areas,plus trees and clonal seed orchards for sandal (Santalum album L.).In: Srimathi RA, Venkateshan KR, Kulkarni HD (eds) Recent ad-vances in research and management of sandal (Santalum album L.)in India. Associated, New Delhi, pp 281–299Srinivasan VV, Sivaramakrishnan VR, Rangaswamy CR,Ananthapadmanabha HS, Shankara Narayana MH (1992) Sandal(Santalum album L.). Institute of Wood Science and Technology(ICFRE), Bangalore, pp 1–60Uniyal DP, Thapliyal RC, Rawat MS (1985) Vegetative propagation ofsandal by root cuttings. Indian Forest 3:145–148

5-Bromo­pyrimidin-2(1H)-one

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