Djoko Agus , Purwanto

M. Rasjad, Indra

Susilowati, Susilowati

Sutini, -

English

UPN Jatim Repository

Qou-uttdoi BIOLOGICAL RESEARCHESBerkiilaPewlilkmHayatiISSN: 08526834|E-ISSN:2337-389XVolume22|No. 1 |December|2016Original ArticleGrowth and accumulation of flavan-3-ol in Camelliasinensisthroughcallusculture and suspensionculture methodSutini Sutini1 *.SusilowatiSusilowati2. M. RasjadIndra’.Djoko AgusPurwanto’1: Departmentof Agrotechnology. Facultyof Agriculture. UniversitasPembangunanNasional’ Departmentof ChemicalEngineering. Facultyof IndustrialTechnology.UniversitasPembangunanNasional Departmentof Physiology. Facultyof Medicine. Brawijaya Universitydepartmentof Pharmaceutical. Facultyof Pharmacy. AirianggaUniversityAbstractThisstudywasaimedto assessflavan-3-ol biomassin C. sinensisthroughcallusculturesandsuspensionculturesderivedfrom leaf explants. Callusinitiation of bothcultureswereusingMurashigeandSkoogmediumwereenrichedwith plant growth regulatorsNaphthalene^\cet ic Acid 3.0 mg/Land kinetin 2.0 mg/L. The proceduresin thisstudywere: ( 1 ) callus initiation by cutting the leavesof C. sinensisshootsthen plantedon MurashigeandSkoogmediumthat wereenrichedwith plant growth regulators. ( 2) subcalluscultureon fresh mediumthatenrichedwith thesamegrowth regu-lators. (3) suspensionculture initiation of liquid callus. (4) growth examinationof callusand suspensionculturesin week 12. (5) e xaminationofqualitative-quantitativecontentof flavan-3-olin suspensionculturesat week4. Theresultsshowthat suspensionculturescontainbiomassflavan-3- olthat increasein thesamemannerof the increaseof callusageandweightKey words: biomassafiavan-3-ol. C.sinensis. MurashigeandSkoog.naphthaleneaceticacid, kinetin.Received: 31 August2015Revised: 26January2017Published: 26 February2017IntroductionFlavan-3-ol is one of the secondarymetabolitescontainedin C. sinensis, which can be utilized in variousfields including medical industry. This compoundis clas-sified into phenolgroupwhich can performsanti-oxidantactivity (Giri ct al., 2012; Vinha et al., 2013; El-kassas2014; Khalaf et al., 2008; Makanjuola et al., 2015). Inagricultural industry, flavan-3-ol monomerin the form ofcatcchinscan be used as allelochemical( lndcijil , 2008;CaslcllsE. 2008). It also trimethyl xanthina is utilized infood industriesto supportsfood and beveragebecomingfunctional (Sutini et al., 2011; 2014; 2016; Ferruzzi.2012; Astri, 2009; Ivan, 2005).Flavan-3-ol can be harvestedfrom C. sinensisplantin disturbed farming area after 3-5 yearsof plantationwith special treatments( Ryo. 2009; Asri, 2010). In vitroculture methodhasbeenused widely for variouspurposesuchassecondarymetabolitesproduction, obtainingiden-tic cloneof C. sinensis, obtainingJatrophathat is resistantto drought, and obtainingsugarcanewith abundantyield(Sumaryonoet al., 2005; Sandalet al., 2005; Mochamadet al., 2012; PTPN. 2014). Callus cultures form wascrumband varies, so it shouldbe kept in optimum condi-tion that correspondsto researchpurposes. Productionofflavan-3-ol through in vitro culture (callus culturesandsuspension) has several advantages, namely less timeconsumingcompareto field production, more efficientCorrespondingAuthor:SutiniDepartment of Agrotechnology, Faculty of Agriculture.UniversitasPembangunanNasional. Veteran.EastJava. Surabaya60115. Indonesiatelp :+628123503771e-mail : lien basukitoyahoo.comV( laboratory scale results that can provide industrialneeds), andto freefrom climaticchangeeffect. Therefore,this studywaspurposedto evaluatethe biomassof flavan-3-ol in callus cultures and suspensioncultures derivedfrom leafexplantof C. sinensis.MethodsInitiation of CallusInitiation of callus was done by cutting C. sinensisleaves then planted on Murashige and Skoog medium( MS) enrichedwith plant growth regulatorsNaphthaleneAcetic Acid (NAA ) and kinetin ( Aljabari et al., 2014;Farzanaet al.. 2011; Nikolaevaet al., 2009; OriharaandFuruya, 1990). C. sinensis leaves were washed withrunning water for 30 minutes, and then dipped in mixsolution of fungicide-bactericide3% and 5% of calciumhypochlorite. The leavesthen were rinsed with distilledwater and soakedin ascorbicacid 3% for 15 minutesinculturetubes. Sterilizationwasdoneby soakingtheleavesin 5% of sodium chlorine solution for 30 minutes thenrinsedthreelimes with steriledistilled water. Leaveswerecut in sterileareafor about l cm with forceps. Leavecutswere initiated in solid mediumMS enrichedwith NAA 3mg/L kinetin and 2 mg/L at laminar air flow cabinet. Atlast, they werestoredin temperature20-25 ° C (Sutini etal., 2008, 2012).CallusSubcultureCalluseswere cut into 2-4 piecessub-callus usingforcepsand needles. The sub-calluseswere subsequentlytransferredto the fresh mediumthat hassimilar composi-tion asinitiation medium. Sub-calluseswerestoredat thesametemperatureas previousstepor 20-25UC (Sutini etal., 2008, 2012).in in  dk tli > i .t >r ^*i l l bpii ibt 22 I 20lf »(iPublishedb) i PBlEasi.laxa Open Access d wuw hcrkaluhavaliora27Qou-uttdoi BIOLOGICAL RESEARCHESBerkiila Pewlilkm HayatiISSN: 08526834|E-ISSN:2337-389XVolume 22 | No. 1 | December | 2016Original ArticleGrowth and accumulation of flavan-3-ol in Camellia sinensis throughcallus culture and suspension culture methodSutini Sutini 1 *. Susilowati Susilowati 2. M. Rasjad Indra '. Djoko Agus Purwanto'1: Department of Agrotechnology. Faculty of Agriculture. Universitas Pembangunan Nasional' Department of Chemical Engineering. Faculty of Industrial Technology. Universitas Pembangunan Nasional‘Department of Physiology. Faculty of Medicine. Brawijaya Universitydepartment of Pharmaceutical. Faculty of Pharmacy. Airiangga UniversityAbstractThis study was aimed to assess flavan-3-ol biomass in C. sinensis through callus cultures and suspension cultures derived from leaf explants. Callusinitiation of both cultures were using Murashige and Skoog medium were enriched with plant growth regulators Naphthalene^\cet ic Acid 3.0 mg/Land kinetin 2.0 mg/L. The procedures in this study were: ( 1 ) callus initiation by cutting the leaves of C. sinensis shoots then planted on Murashigeand Skoog medium that were enriched with plant growth regulators. ( 2) sub callus culture on fresh medium that enriched with the same growth regu-lators. (3 ) suspension culture initiation of liquid callus. (4) growth examination of callus and suspension cultures in week 12. (5) e xamination ofqualitative-quantitative content of flavan-3-olin suspension cultures at week 4. The results show that suspension cultures contain biomass flavan-3- olthat increase in the same manner of the increase of callus age and weightKey words: biomassa fiavan-3-ol. C. sinensis. Murashige and Skoog. naphthalene acetic acid, kinetin.Received: 31 August 2015 Revised: 26 January 2017 Published: 26 February 2017IntroductionFlavan-3-ol is one of the secondary metabolitescontained in C. sinensis, which can be utilized in variousfields including medical industry. This compound is clas-sified into phenol group which can performs anti-oxidantactivity (Giri ct al., 2012; Vinha et al., 2013; El-kassas2014; Khalaf et al., 2008; Makanjuola et al., 2015). Inagricultural industry, flavan-3-ol monomer in the form ofcatcchins can be used as allelochemical ( lndcijil, 2008;Caslclls E. 2008). It also trimethyl xanthina is utilized infood industries to supports food and beverage becomingfunctional (Sutini et al., 2011; 2014; 2016; Ferruzzi.2012; Astri, 2009; Ivan, 2005).Flavan-3-ol can be harvested from C. sinensis plantin disturbed farming area after 3-5 years of plantationwith special treatments ( Ryo. 2009; Asri, 2010). In vitroculture method has been used widely for various purposesuch as secondary metabolites production, obtaining iden-tic clone of C. sinensis, obtaining Jatropha that is resistantto drought, and obtaining sugar cane with abundant yield(Sumaryono et al., 2005; Sandal et al., 2005; Mochamadet al., 2012; PTPN. 2014). Callus cultures form wascrumb and varies, so it should be kept in optimum condi-tion that corresponds to research purposes. Production offlavan-3-ol through in vitro culture (callus cultures andsuspension ) has several advantages, namely less timeconsuming compare to field production, more efficient• Corresponding Author:SutiniDepartment of Agrotechnology, Faculty of Agriculture.Universitas Pembangunan Nasional. Veteran.East Java. Surabaya 60115. Indonesiatelp :+628123503771e-mail : lien basukitoyahoo.comV( laboratory scale results that can provide industrialneeds), and to free from climatic change effect. Therefore,this study was purposed to evaluate the biomass of flavan-3-ol in callus cultures and suspension cultures derivedfrom leaf explant of C. sinensis.MethodsInitiation of CallusInitiation of callus was done by cutting C. sinensisleaves then planted on Murashige and Skoog medium( MS) enriched with plant growth regulators NaphthaleneAcetic Acid (NAA) and kinetin ( Aljabari et al., 2014;Farzana et al.. 2011; Nikolaeva et al., 2009; Orihara andFuruya, 1990). C. sinensis leaves were washed withrunning water for 30 minutes, and then dipped in mixsolution of fungicide-bactericide 3% and 5% of calciumhypochlorite. The leaves then were rinsed with distilledwater and soaked in ascorbic acid 3% for 15 minutes inculture tubes. Sterilization was done by soaking the leavesin 5% of sodium chlorine solution for 30 minutes thenrinsed three limes with sterile distilled water. Leaves werecut in sterile area for about l cm with forceps. Leave cutswere initiated in solid medium MS enriched with NAA 3mg/L kinetin and 2 mg/L at laminar air flow cabinet. Atlast, they were stored in temperature 20-25 °C (Sutini etal., 2008, 2012).Callus SubcultureCalluses were cut into 2-4 pieces sub-callus usingforceps and needles. The sub-calluses were subsequentlytransferred to the fresh medium that has similar composi-tion as initiation medium. Sub-calluses were stored at thesame temperature as previous step or 20-25 UC (Sutini etal., 2008, 2012).in in • dk tli > i . t >r ^* ill bpiiibt 22 I 20 lf»(iPublished b) i PBlEasi .laxa Open Access d wuw hcrkaluhavali ora27

Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

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Growth and accumulation of flavan-3-ol in Camellia sinensis through callus culture and suspension culture method

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