Study On Selection Of Ex-vivo Research Materials And Methods In Inducing Formation Of Sesquiterpene Compounds In Santalum Album L.

Since the scientists from South China Botanical Garden introduced sandalwood seeds into China in 1962,they have gradually resolved many technical problems including seed storage,germination,raising seedlings and host selection.So far,Santalum album has been planted in large scales in China,especially in Guangdong,Yunnan,Hainan,Fujian and Guangxi.The economic value of sandalwood depends on its oil-bearing heartwood.However,the tree begins to form heartwood after 6 years of age.For the moment,the rotation of sandalwood is usually 13-15 years,which means a long investment recovery period.So exploring effective methods to induce heartwood formation at a young age has great significances in promoting the planting of S.album.Sandalwood tree is so valuable it is important to explore alternative research systems,which can be used to select effective exogenous substances before tested in standing trees.Besides,alternative research systems,for example,callus if workable,will be conducive to molecular studies relative to sandalwood heartwood formation.This study focuses on the effects of exogenous substances on the biosynthesis of sandalwood major sesquiterpene compounds,main purposes including:(1)to looking for alternative materials for the selection of exogenous substances,(2)to obtain effective methods for promoting heartwood formation of S.album.Results were as follows:1.Sandalwood branches showed promise to be alternative materials for exogenous substance study.Leaves,branches(ca.1 cm in diameter,2 years old)and embryogenic mass were used as materials,from which volatile oil was extracted by petroleum ether(hydro distillation was also used for leaves)and subject to gas chromatography-mass spectrometry(GC-MS)analysis.Results showed that the volatile oil(extracted by petroleum ether and hydro distillation)of sandalwood leaves contained no detectable desired sesquiterpene compounds and much pigment and other substances,so leaves should not be used as alternative materials.Sandalwood branches containing desired sesquiterpene alcohols were expected as alternative materials.2.Efficient and reliable media for callus induction and proliferation were established.Callus originated from stem nodals was effectively induced on MS + Thidiazuron(TDZ 0.2-1.0 mg/l),with induction rates ranging from 82% to 89%.And the resulting callus was bright yellow,nodular and compact.For callus proliferation,300 mg of fresh callus having cultured for 7-10 d on the 2nd subculture was cut into small pieces and transferred to 25 ml of liquid medium(MS + TDZ 1.0 mg/l)in 100 ml Erlenmeyer flasks.Callus was subcultured once in 7-10 d by discarding the old medium and adding fresh medium.After a month of culture,the fresh weight of callus could reach 6-10 g,which was sufficient for detection of sesquiterpene components.3.Live Colletotrichum gloeosporioides(The Genebank accession number: MF070491)can promote the synthesis and accumulation of sesquiterpene components in sandalwood callus,which showed that these components may play an important role in defense.Uninfected callus contained no detectable desired sesquiterpenes,while the callus infected with some fungi showed the presence of desired sesquiterpenes,which suggested that these sesquiterpenes may play an important role in defense.4.Biosynthesis of major sesquiterpene components in S.album heartwood can be induced by trunk infusion of 6-benzyladenine(BA)and hydrogen peroxide.Among the tested two concentrations(2 m M and 4 m M)of five substances(BA,ethylene,methyl jasmonate,hydrogen peroxide,calcium chloride),4 m M BA can effectively promote the longitudinal expansion of induced heartwood.The height of induced heartwood reached 150 cm treated with 4 m M BA four times a year,far higher than other treatments and the control.The oil contents of induced heartwood by 4 m M BA and 4 m M hydrogen peroxide were higher than others,being 2.75% and 2.85%,respectively,4.2 and 4.4 times higher than the control(0.65%).For oil quality,essential oil extracted from heartwood induced by hydrogen peroxide(2 m M and 4 m M),2 m M ethylene or 4 m M calcium chloride met the international standards.Relative content of α-santalol in essential oil from induced heartwood by 4 m M BA or 2 m M methyl jasmonate was a little lower but close to the international standards.Considering the longitudinal extension of induced heartwood and its oil content and quality,Trunk infusion of 4 m M BA can be used for promoting S.album heartwood formation at a young age.