In vitro microrhizome induction is considered as an effective tool for high yielding rhizomatous crops. In this study, the

effect of sucrose and BAP was examined to establish a suitable protocol for in vitro microrhizome production of Zingiber

officinale Rosc. ‘Tambunan’. The in vitro derived plantlets were used as explants and cultured on Murashige and Skoog

(MS) medium treated with a combination of sucrose and BAP at various concentrations and maintained at 25 ± 2°C with 16

hr of photoperiod. After three months of culture, explant responded well on MS medium supplemented with 60 g/L of

sucrose and 6 mg/L of BAP compared to other treatments. This treatment had significantly promoted the highest number of

microrhizomes (seven) with a total weight of 2.90 g and a total number of 35 buds. Acclimatization of this microrhizome

showed 88% of survivability rate after 21 days with a formation of new shoot and root. The current finding revealed the

potential of microrhizomes for large-scale production of healthy planting material to support the ginger industry in this region

Devina David,

Jualang Azlan Gansau,

Ling, Ee Chuwan

UKM Journal Article Repository

* To whom correspondence should be addressed.Malays. Appl. Biol. (2018) 47(6): 47–52OPTIMIZING SUCROSE AND BAP CONCENTRATIONS FORIN VITRO MICRORHIZOME INDUCTION OFZingiber officinale Rosc. ‘Tambunan’DEVINA DAVID1*, LING EE CHUWAN1 and JUALANG AZLAN GANSAU21Faculty of Sustainable Agriculture, Universiti Malaysia Sabah, 90509 Sandakan, Malaysia2Faculty of Science and Natural Resources, Universiti Malaysia Sabah, 88400 Kota Kinabalu, Malaysia*E-mail: ddevina@ums.edu.myAccepted 20 December 2018, Published online 31 December 2018ABSTRACTIn vitro microrhizome induction is considered as an effective tool for high yielding rhizomatous crops. In this study, theeffect of sucrose and BAP was examined to establish a suitable protocol for in vitro microrhizome production of Zingiberofficinale Rosc. ‘Tambunan’. The in vitro derived plantlets were used as explants and cultured on Murashige and Skoog(MS) medium treated with a combination of sucrose and BAP at various concentrations and maintained at 25 ± 2°C with 16hr of photoperiod. After three months of culture, explant responded well on MS medium supplemented with 60 g/L ofsucrose and 6 mg/L of BAP compared to other treatments. This treatment had significantly promoted the highest number ofmicrorhizomes (seven) with a total weight of 2.90 g and a total number of 35 buds. Acclimatization of this microrhizomeshowed 88% of survivability rate after 21 days with a formation of new shoot and root. The current finding revealed thepotential of microrhizomes for large-scale production of healthy planting material to support the ginger industry in this region.Key words: 6-Benzyladenine (BAP), ginger, microrhizome, sucrose, ZingiberaceaeINTRODUCTIONGinger, also botanically known as Zingiberofficinale Roscoe, is one of the world mostimportant and value plant that consumed as adelicacy, medicine and spices. In Malaysia, gingeris cultivated commercially in Pahang, Johor,Selangor, Sabah and Sarawak (Suhaimi et al., 2012).Zingiber officinale Rosc. ‘Tambunan’ is a popularcultivar of ginger in Sabah and it is mainlycultivated in Tambunan and Keningau areas. Locallyknown as Tambunan ginger, it was believed that thiscultivar was originally from Bentong, Pahang.However, application of biotechnology approachessuch as metabolic fingerprinting (Mahdi et al., 2010)and microsatellite DNA (Mahdi et al., 2013) ableto differentiate between Malaysian ginger cultivarsincluding Bukit Tinggi, Tanjung Sepat and Sabahcultivars. Zingiber officinale Rosc. ‘Tambunan’ hasgained its popularity for its flavor and aroma, thuscontribute to high market demand. However, theplanting areas and production of gingers in Sabahdeclined from 467 hectares (5,848.4 tonnes) in2006 to only 197 hectares (1,341.2 tonnes) in 2016(Department of Agriculture Sabah 2006; 2016) andit was due to attack of soil-borne bacterial wiltdisease that caused by Ralstonia solanacearum(Cosmas et al., 2016). This incident had caused asignificant decrease in ginger yield as well as theproduction of ginger rhizomes as planting materials.Microrhizome is a miniature rhizome inducedvia in vitro condition and possesses similarcharacteristics as normal rhizome in anatomicalfeatures such as starch grains, well-developed oilcells and fibers (Maiti & Yepthomi, 2015). Thetechnique of in vitro microrhizome induction hasbeen identified as a solution to supply high qualityplanting materials especially for rhizomatouscrops (Archana et al., 2013; Nayak & Naik, 2006).Besides, it has advantages of commercial valuewhere it is easier to transport, store and can be usedin germplasm conservation (Chirangini & Sharma,2005). The induction of in vitro microrhizome ofmany Zingiberaceae species was reported to beinfluenced by several factors such as photoperiod(Abbas et al., 2014; Archana et al., 2015;Swarnathilaka et al., 2016), plant growth regulators48 IN VITRO MICRORHIZOME INDUCTION OF Zingiber officinale Rosc. ‘Tambunan’(Rout et al., 2001; Archana et al., 2013), nutrientcomposition (Zheng et al., 2008) and culture vessel(Singh et al., 2014). Previously, a protocol for invitro mass propagation of Z. officinale Rosc.‘Tambunan’ was successfully established usingrhizome bud as explant (David et al., 2016).Therefore this study was conducted to determine andoptimize the sucrose and 6-Benzyladenine (BAP)concentrations for in vitro microrhizome productionof this ginger cultivar.MATERIALS AND METHODSPlant materialFour months old of in vitro derived plantletsof Zingiber officinale Rosc. ‘Tambunan’ from theprevious study (David et al., 2016) were selectedand used as explants. Each explant was excised into4 cm height and the root was trimmed into a shorterlength. The excised explants were initially culturedin a culture jar containing 30 mL of MS (Murashige& Skoog, 1962) medium without supplementingany growth regulator for two weeks to minimizecarryover effect of growth regulators in previoustreatments.Culture treatmentExplants were then transferred to culture jarcontaining 30 mL of MS medium supplementedwith combinations of sucrose and BAP at variousconcentrations. Three levels of sucrose concentra-tions (30, 60 or 90 g/L) and two concentrations ofBAP (6 or 9 mg/L) were tested in this study witha total of six combination treatments. Mediumwithout any addition of sucrose and BAP was servedas control. The pH of the culture media was adjustedto 5.7 before solidifying with 10 g/L of agar andsterilized at 121°C for 20 min. All cultures weremaintained at the temperature of 25 ± 2°C with 16hr photoperiod.Data collection and statistical analysisAll cultures were observed periodically for 12weeks. Each treatment consisted of ten replicatesof one explant each, arranged in CompletelyRandomized Design. Data were collected for twomain parameters (i) vegetative growth of explantwhich include the number of leaves, roots, shoot andshoot length (cm); (ii) microrhizome inductionwhich includes a number of microrhizome, freshweight of microrhizome (g), number of buds andnumber of buds per microrhizome. Data wereanalyzed using one-way analysis of variance(ANOVA) through IBM SPSS Statistic Version 25.0New York, IBM Corp. The significant differencebetween the means was separated by Tukey’sHonestly Significant Different (HSD) test at p < 0.05.AcclimatizationA total of 20 in vitro derived microrhizomeswere selected for establishment in the nursery toinvestigate their ability to acclimatize in ex vitrocondition. For this purpose, microrhizomes weretreated with 0.5% (w/v) of fungicide for 20 minbefore planting in pots containing coco peat. Themicrorhizomes were initially covered with apolythene sheet for two weeks to maintain highhumidity and irrigation was done through mistingonce a day with tap water. Growth performance ofmicrorhizome was observed and the survival ratewas recorded.RESULTS AND DISCUSSIONEffect of BAP and sucrose on vegetative growthof explantThe vegetative growth of ginger explant wasobserved and shown in Figure 1. It was observed thatafter one week of culture, whitish buds were initiatedfrom the explant (Figure 1A) and later changed itscolor to pale green before developing into shootswithin 15 days of culture. The shoots continued tolengthen and then started to form leaf sheaths. Theleaf sheaths grew into flat leafy structure after sixweeks of culture and the well-developed gingerplantlets with leaves and roots were obtained within12 weeks of culture (Figure 1B).Supplementation of sucrose and BAP in MSmedium had significantly influenced all vegetativegrowth parameters of Z. officinale Rosc. ‘Tambunan’explants. Combination treatments including 30 g/Lsucrose + 6 mg/L BAP; 30 g/L sucrose + 9 mg/LBAP and 60g/L sucrose + 6 mg/L BAP significantlyproduced the maximum number of leaves (28 - 41)after three months of culture (Figure 2A). Treatmentsof 30 g/L sucrose + 6 mg/L BAP and 60 g/L sucrose+ 6mg/L BAP also induced 55 and 53 of new roots,respectively (Figure 2B). The combinations ofsucrose at 30 and 60 g/L with 6 and 9 mg/L of BAPenhanced the production of a new shoot, however,increased in sucrose concentration (90 g/L),decreased shoot multiplication as well as shootlength of the explants (Figure 2C and 2D). Thisevent was observed similar in leaves and rootproduction and this finding was in agreement witha study by Kusumastuti et al. (2014) that 30 g/L ofsucrose was beneficial in promoting shoots androots multiplication of Z. aromaticum as comparedto higher sucrose concentration (60 and 90 g/L).Sucrose is the most common carbon source and actsas an energy source to maintain osmotic potentialas well as ensuring optimal development during invitro growth of plants (Yaseen et al., 2013).IN VITRO MICRORHIZOME INDUCTION OF Zingiber officinale Rosc. ‘Tambunan’ 49Fig. 1. In vitro growth and development of microrhizomes in Zingiber officinale Rosc.‘Tambunan’. (A) Initiation of whitish bud after one week of culture (B) Well developedplantets with leaves and roots after 12 weeks of culture (C) Bulging of pseudostem indicatemicrorhizome induction (D) Swollen of basal roots from the microrhizome (E)Microrhizomes produced in treatment of 60 g/L sucrose and 6 mg/L BAP (F) Budsemergence from acclimatized microrhizome (G) Acclimatized microrhizomes with shootsand roots after 14 days of transplant. Bar (A) 0.5 cm; (B - G) 1.0 cm.Effect of BAP and sucrose on in vitro induction ofmicrorhizomeThe initial indication of microrhizomeinduction was observed when the pseudostem andbasal root of ginger plantlets were swollen (Figure1C and 1D). In this study, the combination ofsucrose with BAP in culture medium hadsignificantly influenced in vitro microrhizomeformation in this ginger. Explants cultured on MSmedium treated with 60 g/L of sucrose and 6 mg/Lof BAP had significantly produced an average ofseven microrhizomes with a total weight of 2.90 gand a total number of 35 buds after 12 weeks ofculture (Figure 2E, 2F and 2G). The microrhizomeformed through this treatment was characterizedas a yellow-orange mini rhizome (Figure 1E), andproduced a fresh ginger smell when crushed. Thecurrent finding was in agreement with Maiti andYepthomi (2015), that microrhizome possesses thesame characteristics as the normal ginger rhizome.50 IN VITRO MICRORHIZOME INDUCTION OF Zingiber officinale Rosc. ‘Tambunan’Fig. 2. Effect of sucrose and BAP at various concentrations on in vitro growth and development of microrhizome on Z.officinale Rosc. ‘Tambunan’. (A) Leaf number; (B) Root number; (C) Shoot number; (D) Shoot length; (E) Microrhizomenumber; (F) Microrhizome fresh weight; (G) Bud number; (H) Bud per microrhizome. Notes: Value are expressed as themean of ten replicates ± S.E. Mean values followed by different letters are significantly different at p <0.05. S: Sucrose(g/L); B: BAP (mg/L).IN VITRO MICRORHIZOME INDUCTION OF Zingiber officinale Rosc. ‘Tambunan’ 51In this study, 60 g/L of sucrose was favorable inpromoting microrhizome production might beattributed to high carbon energy in sucrose sincerhizomes mainly contain carbohydrate andsucrose (Nayak & Naik, 2006). Several studies haddemonstrated the effectiveness of sucrose between60–90 g/L on in vitro induction of ginger micro-rhizome (Rout et al., 2001; Zheng et al., 2008;Abbas et al., 2014; Singh et al., 2014). However,the application of 90 g/L of sucrose in the currentstudy showed a detrimental effect. Higher sucrosecontent might induce osmotic stress in this mediumand resulted to slow absorption of nutrient asdescribed previously in Kusumastuti et al. (2014).Acclimatization of microrhizomeA total of 20 harvested microrhizomes weretransplanted to a pot containing cocopeat as mediumand maintained under a high level of relativehumidity. After seven days of transplant, theemergence of buds was observed from the micro-rhizomes (Figure 1F) and later it developed into theshoot. The appearance of root was observed laterafter 14 days of transplant (Figure 1G). New shootcontinues to grow from the microrhizome and thesurvival rate was recorded up to 88.30% after 30days of transplanting. A study by Archana et al.(2013) showed that ginger microrhizomes transferredout to a nursery survived at 90–100% of survivalrate.CONCLUSIONThis study has successfully established an efficientprotocol for in vitro microrhizome induction ofZ. officinale Rosc. ‘Tambunan’. This approachcould be an alternative to solve the issue in lackingginger rhizome as planting material for cultivationas well as for consumption. The current findingrevealed the highest number of microrhizomeformation was observed on treatment of MS mediumsupplemented with 60 g/L of sucrose and 6 mg/L ofBAP. Further study is recommended to examineother factors such as photoperiod, culture media andtypes of plant growth regulators that might affect thein vitro microrhizome induction for this Tambunanginger cultivar.ACKNOWLEDGMENTSThis study was supported by the Faculty ofSustainable Agriculture, Universiti Malaysia Sabah(UMS). The authors would like to acknowledgeMr. Poulus bin Empah from Pertubuhan PeladangKawasan (PPK) Tambunan for providing plantmaterials for this ginger research.REFERENCESAbbas, M.A.U., Taha, H. & Gaber, E.S. 2014. In vitroproduction of microrhizomes in ginger (Zingiberofficinale Roscoe). Journal of Microbiology,Biotechnology and Food Sciences, 4(2): 142-148.Archana, C.P., Geetha, S.P. & Balachandran, I. 2013.Microrhizome and minirhizome production inthree high yielding cultivars of ginger (Zingiberofficinale Rosc.). International Journal ofCurrent Microbiology and Applied Sciences,2(10): 477-484.Archana, C.P., Geetha, S.P. & Balachandran, I. 2015.Effect of ammonium nitrate and photoperiod onin vitro microrhizome induction in three highyielding cultivars of Curcuma longa L. andtheir comparative phytochemical analysis. AsianJournal of Pharmaceutical Science & Techno-logy, 5(1): 45-49.Chirangini, P. & Sharma, G.J. 2005. In vitropropagation and microrhizome induction inZingiber cassumunar (Roxb.) an antioxidant-rich medicinal plant. 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Optimizing sucrose and BAP concentrations for in vitro microrhizome induction of Zingiber officinale Rosc. ‘Tambunan’

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Optimizing sucrose and BAP concentrations for in vitro microrhizome induction of Zingiber officinale Rosc. ‘Tambunan’

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