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IntroductionIndonesia is one of the countries in the

world with the biggest source of biodiversityof orchids, which is approximately 5000 species(Irawati, 2001). Some species of orchids havebeen used as ornamental plants because ofits high aesthetic value (Yulia and Ruseani,2008). The tiger orchid Grammatophyllumscriptum (Lindl.) BI. is one of the best orchidswith high aesthetic value due to the numberof flowers per stack (25–50 flowers); longlasting and vigor, which makes them verygood to be an ornamental plant (Hani, 2015).

Propagation of wild orchids like G.scriptum is needed for conservation andcultivation. Natural orchid propagation hasa low success rate. According to Hani (2015),the environment has a significant role inpropagating orchids naturally in their habitat.The environment comprises moisture,

temperature, and light, along with thestructure of orchid seeds that impede naturalgermination. A microscopic­sized orchid seedconsists of two parts, which are embryo andtesta (seed coat) (Soeryowinoto, 1987). Mostof orchid seeds do not have endosperm, so italso impedes the growth and developmentof the orchid embryo in nature (Hani, 2015).

In vitro culture is one of the maintechniques to propagate orchids. With thisculture, orchids can be propagated easily andquickly (Rosdiana, 2010). The orchid plantletswill face environmental stress when they aretransferred to the ex vitro environment, so ithas a higher risk of death. This happensbecause the structures of the plantlets havethin cuticle layer, imperfect vascular tissue,and reduced stomata function (Torres, 1989).In addition, plantlets in in vitro culture areaused to being in heterotrophic condition witha supply of nutrients available in the culturemedium, so the adjustment in the autotrophiccondition in acclimatization process is goingto be difficult. That is why an appropriate invitro propagation technique is needed so theorchid plantlets will have a higher successrate when doing acclimatization.

Indonesian Journal of Biotechnology, December, 2016 Vol. 21, No. 2, pp. 109–116

The Growth Improvement of Grammatophyllum scriptum (Lindl.) Bl.In Vitro Plantlet using Photoautotrophic Micropropagation System

Aries Bagus Sasongko1, Asruwaidah Fatumi1, Ari Indrianto1,*

1Laboratory of Biotechnology, Faculty of Biology, Universitas Gadjah Mada,Jalan Teknika Selatan, Sekip Utara, Yogyakarta 55281, Indonesia

AbstractTo improve the growth of Grammatophyllum scriptum (Lindl.) Bl. in vitro plantlet, a photoautotrophic

micropropagation system (PMS) was developed by growing in vitro plantlet on VW medium with varyingconcentration of sucrose (0, 5, 10, and 20 g/L) and additional carbon dioxide from the air (bottle coveredwith cap or filter). The result showed that the leaf length would increase up to 6.5 cm with PMS and itwould keep growing by the adding of 5 g/L sucrose. Average number of leaves increased by 6.7 strandswith PMS and the addition of sucrose increased the average quantity of leaves up to 7.7 strands. Averagenumber and root length would increase with PMS and would even increase more with 5 g/L sucroseaddition. PMS with 5 g/L sucrose can increase chlorophyll a and b concentration. The number of stomataper unit area in PMS was lower than closed culture. This shows that PMS can increase the growth of G.scriptum in vitro plantlet and the growth increase would be effective if it is combined with sucrose addition.

Keywords: Plant growth, Grammatophyllym scriptum plantlet, photoautotrophic micropropagation system,in vitro, carbon dioxide

*Corresponding author:Ari IndriantoLaboratory of Biotechnology, Faculty of Biology,Universitas Gadjah Mada, Jalan Teknika Selatan,Sekip Utara, Yogyakarta 55281, IndonesiaE­mail: ariindri@ugm.ac.id

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Factors that affect in vitro plant growthare the type and concentration of carbohydrate,mineral nutrient, phytohormones, light,temperature, medium pH, moisture, gasexchange, and contamination. Plantlets asresults of in vitro culture have a low capabilityof photosynthetic rate, so it needs additionalsugar to grow. The lack of gas exchange inconventional in vitro culture causes thereduction in oxygen and carbon dioxide andthe increase ethylene gas accumulation. Thiscondition will impede the growth processaccelerate aging process (Hew and Yong, 2004).

Photoautotrophic micropropagationsystem (PMS) is a micropropagation methodusing media without sugar, vitamin, andamino acid, and only with inorganic materialsonly. This system emphasizes on the CO2contents, light intensity, and relative moisture.The advantages of this method are to reducemicrobial contamination risk and increaseplantlets’ growth by giving light and highamount of CO2 in the bottle (Xiao and Kozai,2006). This method can induce photosyntheticprocess. PMS has been proven to enhance thegrowth of Cymbidium, Phalaenopsis, andEpidendrum orchids (Teixera da Silva et al.,2005). In order to improve the growth of G.scriptum in vitro plantlet, research about theeffects of PMS on G. scriptum in vitro plantletsis needed.

Materials and Methods

Plant material and growth conditionG. scriptum plantlets aged 12 months

and have two leaves were grown in Vacinand Went (VW) medium in accordance withArditti (2008) with variations of sucrose (0,5, 10, and 20 g/L) and variations of bottlecaps, which were aluminium foil and rubbercap with filter based on certain modified pipe(Table 1). The culture was maintained by

giving continuous lighting and roomtemperature 25°C for eight weeks. Apreliminary test on the amount of carbondioxide that got into the culture bottle wasdone in accordance with Haney et al. (2008).

Chlorophyll concentration testChlorophyll concentration in orchid

plantlets was measured at the beginning andthe end of the research in accordance withSpecht et al. (1975). The plantlet leavesweighing 0.1 g were crushed in a porcelainmortar and dissolved in 10 mL of 80% acetone,then filtered with filter paper. The solutionwas put into the test tube and covered withaluminum foil and then absorbance wasmeasured using spectrophotometer with thewavelength of 645 nm and 663 nm. Chlorophyllconcentration was calculated using thefollowing Eq. 1 and 2.

Measurements of growth parameterThe leaves that were counted were those

that had perfect shape and green in color. Allmeasurements were done once a week foreight weeks.

Anatomic preparation of stomata and leavesLeaves of G. scriptum plantlet from each

treatment were taken and polypropylene­based glue was smeared on the abaxial surfaceas the mold. After it dried, the glue was peeledand observed under a microscope with amagnification of 10x10, and then the calculationof the number of stomata with leaf area of 4mm2 was made. The transverse section of theleaves was observed by making new incisionson the third plantlet leaf with a holder in theform of carrot slices to make the incisionseasier. The result was put in the beaker glass

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Table 1. Variation of sucrose concentration in VW medium.

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and observed using a light microscope withmagnification of 4x10. The pictures were takenusing Optilab.

Results and DiscussionThis research uses two systems in vitro

cultures: closed system and open system. Thedifference between these two systems lies inthe air exchange factor between in vitroculture and ex vitro environment. Thedifference between these two systems has animpact on the environment of in vitroculture. The closed system has high moisture,low CO2, and ethylene gas accumulation. Incontrast, the open system has relatively lowmoisture, there was more of CO2 and there

was no accumulation of ethylene.PMS applies the open in vitro culture

system with modification of sucrose­lessmedium. Plantlets grown using this systemwill have a better growth rate than that usingthe closed system. In addition, the survivalrate of in vitro plantlet will increase becauseof its ability to adapt to the ex vitroenvironment.

This research focuses on the growth ofG. scriptum plantlets through in vitrocultivation in closed and open systems bothby PMS medium without sucrose and withsucrose. Growth characters observed werethe leaf organs and roots, along withchlorophyll content.

Figure 1. Morphology of G. scriptum plantlets in the open and closed system with various sucrose concentration.Photoautotrophic micropropagation system, which is an open system with a sucrose­less medium. Sign (+) indicatesopen system/CO2 addition from the air, and sign (­) indicates closed system/no air exchange. The arrow signindicates the height of plantlets in in vitro culture. Scale bar = 5cm.

Figure 2. The growth of leaves and roots organs of G. scriptum plantlets on various treatments: a) number ofleaves; b) leaf length; c) number of roots; d) length of root.

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Growth of leaves and root in G. scriptumplantlets

G. scriptum plantlets grown on mediumwithout sucrose and addition of CO2 showedbetter growth rate than those grown in closedsystem on both media with sucrose andwithout sucrose. Morphologically, plantletsgrown by PMS system and its modificationhas stronger habitus than those grown usingthe closed system (Figure 1). However, thesame effect was also seen after adding 10 g/Lsucrose in the closed system.

CO2 addition in the system affected theleaf growth, especially when compared withclosed culture with sucrose concentration of20 g/L. However, plantlet leaves grown inPMS with an addition of 5 g/L sucrose werelonger when compared with all treatments.In addition to the leaf length, the leaf numberin the plantlets grown on PMS was alsoincreased. It also happened to the leaves onthe plantlets grown in the closed system withan addition of 20 g/L sucrose (Figure 2a and2b). This also shows that the function ofphotosynthesis would be more optimum ifthe size and quantity of leaves increased.

The number of roots in the plantletsgrown in PMS increased. Sucrose additionon PMS (starts from 5–10 g/L) would increasethe average of plantlet roots quantity up to5.7 times. Root growth was also shown bythe root elongation. Plantlets grown in PMSwith the addition of 5–20 g/L sucrose havea better average of root length compared tothe closed system (Figure 2c and 2d). Thisshows that the function of nutrient absorptionby the root will be more effective using PMS.

Leaf anatomyIn addition to the length and number

of leaves, leaf thickness and stomata densitywas also observed. The results showed thatthe leaves of plantlets in the treatment of aclosed system, in general, were thickercompared to PMS. The addition of sugar toPMS had a tendency to increase leaf thicknessfor more than 50% compared to PMS. Leavesappeared to become increasingly thickerwithin a closed system with the addition of20 g/L of sucrose (Table 2). It suggests that

closed system can lead the plantlet leafthickness increased due to the high watercontent in the culture system. This causes thecells to undergo expansion. However, thefactors that led to leaf cell division has notbeen observed in this study.

In ex vitro, G. scriptum, stomata couldbe found on both sides of the leaf, which isboth the adaxial and abaxial side. It is evidentin Figure 3 that the quantity of stomata onthe adaxial section of the leaf is much largerand denser compared to its abaxial section.Stomata open around 7:00 AM in the morningand close at night. It indicates that CO2absorption occurs from morning to evening.However, the precise time on which stomatacloses on G. scriptum has not been investigatedin this study.

The number of stomata in plantlet leavesper 4 mm2 increased in a closed system. Thestomata would then decline in PMS. In aclosed system with high levels of sucrose

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Table 2. The thickness of G. scriptum plantlet cross­section incision on various treatments.

Figure 3. Stomata cross­section on ex vitro G. scriptumleaves: a) adaxial stomata (red circle); b) abaxial stomata;c) abaxial stomata in the morning at 07.00; d) abaxialstomata at night at 19.00.

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(20 g/L), the number of stomata had increasedto almost 30%, or 115 stomata per 4 mm2. Theaddition of CO2 and 10 g/L sucrose loweredthe number of stomata to 94.33 stomata per4 mm2, or as much as 40% less compared withthe closed systems without the addition ofsucrose (Figure 4). This may indicate that thepresence of CO2 in the PMS can reduce thenumber of stomata. It is suspected that thereis a connection between CO2 and the numberof stomata per unit area.

Chlorophyll levelThe quantity of chlorophyll related to

the level of photosynthetic activity.Increased chlorophyll levels mean increasedphotosynthesis activity, and thus can beused as an indicator of growth andeffectiveness of CO2 binding. The totalchlorophyll quantity, or chlorophyll a and bin PMS with additional 10 g/L sucrose,showed the highest results. Without theaddition of sucrose, plantlets grown on PMSshowed an increase in the quantity of totalchlorophyll compared to plantlets in a closedsystem without the addition of sucrose,which quantified to almost 25% (Figure 5).This shows that there is a connectionbetween chlorophyll content and addition ofCO2.

Cultured in vitro orchids have shownlower acclimation success rate. This is due todifferences between the environmental

conditions (ex vitro) at the time ofacclimatization and conditions when theplantlets were cultured in vitro. Suchdifferences include the level of moisture,nutrient supply, temperature, light intensity,and carbon dioxide for photosynthesis. Inthis culture, aseptic conditions must bemaintained in order to prevent contaminationby bacteria or fungi, through sterile conditionsand closed systems. However, this can leadto a shortage of carbon dioxide resulting inreduced effective autotrophic function of theplantlets.

PMS is expected to supply plantletswith carbon dioxide, which in turn willaccustom these plantlets to startphotosynthesising optimally during in vitrobecause they are supported by an adequatesupply of carbon dioxide. Carbon dioxideacts as a raw material in photosynthesis (highcarbon dioxide levels support plant growth).In this system, the plantlets were grown ona medium without sucrose. The process aimsto prioritize the CO2 consumption as a carbonsource for plantlet photosynthesis rather thansucrose. This will result in optimalphotosynthesis during acclimatization whileincreasing the survival of plantlets in ex vitroconditions.

In addition to the increase of carbondioxide from the air, which flows throughthe gooseneck pipe, the plantlets in VWmedium were also given various sucrose

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Figure 4. Comparison of the number of stomata per 4 mm2 area in G. scriptum abaxial plantlets leaves within a closedsystem and PMS, with sucrose variations in VW mediums (for 8 weeks).

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treatments. The plantlets ability tophotosynthesize in in vitro conditions alsodecreased, thus requiring more exogeniccarbon for growth, which could be obtainedfrom sugar or sucrose (Hew and Yong, 2004).This led to the need to understand thecorrelation between carbon dioxide levelsand sucrose quantity, both of which werenutrients needed by the plantlets. In general,20–30 g/L of sucrose was added to every literof the medium. With the addition of morecarbon dioxide, it was hoped that the amountof nutrient given to each growth mediumcould be decreased to increase the plantletsautotrophic attribute. The increase in carbondioxide levels should be proportional to theincrease in the plantlets growth parameter.The study measured the growth parametersof G. scriptum orchids, which were leafquantity, leaf length, root quantity, and rootlength.

A large number of leaves in the PMStreatment with the addition of 20 g/L sucroseshowed that there was a large sugar sinkcapacity within G. scriptum plantlets.According to Campbell et al. (2004), plantshave organs that function as sugar sink andsugar source. Organs that function as sugarsinks are roots, tubers, stems, fruits, and leavesin their development phases (Campbell et al.2004). Therefore, as plantlets receive a largesugar supply, it can be then stored in the stem.

It is similar to fully developed leaves and itindicates that sugar levels can increase thenumber of leaves in the plantlets.

The roots are essential duringacclimatization. For this reason, duringacclimatisation plantlets that are chosenshould have strong roots in order to adapt toex vitro environments and can quickly absorbwater and other nutrients content in theacclimatization medium. The existence oflong and abundant roots can increase a plantability to absorb nutrients. This is especiallytrue for epiphytic plants like the G. scriptum.

Photosynthesis process in plants is notonly affected by light, water, carbon dioxidelevels and other factors but it is also greatlyinfluenced by the chlorophyll within theplants themselves (Sasmitamihardja andSiregar 1997). Chlorophyll is a green plantpigment and is located in the chloroplasts.The ratio of chlorophyll a and chlorophyll bin higher plants is 3:1 (Suprapti, 2005). Thisstudy measured the levels of chlorophyll a,chlorophyll b, and the total chlorophyll amountwithin plantlets in each individual treatments.High chlorophyll levels indicate that the plantsare actively photosynthesizing and can alsobe applied as a growth indicator.

This study also observed G. scriptumanatomically by examining leaf stomata andleaf thickness by measuring its cross­section.Orchid leaves are generally divided into two

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Figure 5. Average concentration of chlorophyll a, b and the total (mg/g) in G. scriptum plantlets within a closedsystems and PMS, with sucrose variations in VW mediums (for 8 weeks).

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types, thin and succulent (Hew and Yong,2004). Leaf thickness is associated withphotosynthetic activity. Thin­leaved orchidsgenerally have type C3 photosynthesis, whilethe thick­leaved variant have CAM(Crassulacean Acid Metabolism). Apart fromthe thickness of the leaves, photosynthesistypes can also be seen from the time of stomatalopening. C3­type plants’ stomata will openat noon, while CAM plants’ opens at night.This is a form of environmental adaptationto temperature and water availability (Zotsand Tyree, 1996). CAM plants are usuallyplants that can adapt in dry places. Cropyielded from in vitro culture generally havethick leaves. Differences in the thickness ofthe leaves can be affected by the division ofparenchyma cells in the mesophyll. It can alsobe caused by parenchyma cells increasing insize on mesophyll due to high moisturecondition. Moisture or atmospheric watercontent is an important factor that affects thesuccess rate of acclimatization. In vitro cultureplantlets commonly have thick leaves becausethe plantlets grew on a medium with highhumidity. This is due to the extremeenvironment switch, which resulted lowacclimatisation success rate. Thinner leaves,on the other hand, are assumed to be moreadaptable in environments with low humidity.PMS treatment during the in vitro cultureprocess was known to have lower air moisturearound plantlets due to the increased aircirculation inside the culture bottle that flowsthrough the gooseneck pipe opening.

Stomata were found on both the adaxialand abaxial sides of the leaf, making the leafbelong to amfistomatic. However, it is evidentin Figure 3b that the number of stomata onthe abaxial leaves is much larger and denserthan those of abaxial leaves (Figure 3a). Figure3c and 3d shows that G. scriptum leaf stomataopens in the morning at around 07.00 andcloses at 19.00 in the evening. This provedthat G. scriptum has type C3 photosynthesisbecause the stomata open in the morning,thus carbon dioxide fixation took place in themorning.

Stomata has become an important objectof observation because the majority of moisture

within a plant will exit through the structure(Zots and Tyree, 1996). Stomata is also a highlyadaptive structure. Desert plants have a lowerstomata density compared to the plants thatgrow in wetter environments. Campbell et al.(2004) stated stomata density is flexible andchanges accordingly depending on thetreatment given. Addition of light intensityand low carbon dioxide levels can increasestomata density (Campbell et al., 2004).

ConclusionsThe use of PMS or an open system with

additional CO2 from the air and sugar­freemedium can increase the growth of G. scriptumplantlets. PMS provides better results in termsof morphology, anatomy, and physiology,increases leaf and root growth, lower stomatacount, thicker leaves, and higher chlorophylllevels.

AcknowledgmentsThe researchers are thankful for the

support from Universitas Gadjah MadaResearch Directorate through the ProgranPeningkatan Kapasitas Peneliti Dosen Mudadengan Sumber Dana Bantuan PendanaanPerguruan Tinggi Negeri Badan HukumDirektorat Penelitian (Capacity Enhancementfor Young Lecturer Researchers Fundedthrough Financial Assistance for AutonomousPublic Universities from the ResearchDirectorate) number: 1590/UN1­P.IIILT/DIT­LIT/2016 dated April 28, 2016.

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