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Top grafting performance of some cocoa clones as affected by scion budwood number

163PELITA PERKEBUNAN, Volume 31, Number 3, December 2015 Edition

Pelita Perkebunan 31(3) 2015, 163—174

Top Grafting Performance of Some Cocoa (Theobroma cacao L.)Clones as Affected by Scion Budwood Number

Fakhrusy Zakariyya1*) and Fitria Yuliasmara1)

1)Indonesian Coffee and Cocoa Research Institute, Jl. PB. Sudirman 90, Jember Indonesia*)Corresponding author: [email protected]

Received: 13 November 2015 / Accepted: 27 November 2015

Abstract

Reducing budwood number is an efficient effort to overcome problemsrelated with limited scion materials. The objective of this research was to studythe effect of scion budwood number in some clones on the performance of graftedcocoa seedlings. The research was conducted at Kaliwining Research Station,Indonesian Coffee and Cocoa Research Institute, Jember, Indonesia at an elevationof 48 m above sea level. Layout for this study used factorial with 2 factors inrandomized complete block design, with four replications for every treatment.The first factor was clone type, namely MCC 02 and Sulawesi 1; whereas the secondfactor was number of grafted scion budwood, namely one, two, and three graftedbudwoods. There was no interaction between clone and number of scion budwoodfor variables of shoot length, stem girth, content of total chlorophyll, chlorophylla, and chlorophyll b. Meanwhile, there was interaction for stomatal conductanceand stomatal diffusion resistance. Clone significantly affected photosynthesisand stomatal diffusion resistance, while number of scion budwood affected signifi-cantly the shoot length. Photosynthesis activity of MCC 02 was higher comparedto Sulawesi 1. In average, stomatal diffusion resistance of Sulawesi 1 was higherthan MCC 02. The shoot length of one grafted budwood was higher than thetwo or three grafted budwood.

Keywords: top grafting, budwood, clones, cocoa, Theobroma cacao, photosynthesis

INTRODUCTION

Top grafting is the most common methodused in cocoa plant propagation which isaimed to obtain a superior trait combinationbetween two parents by clonal method. Prin-cipally, that method combines scion(budwood) with rootstock which merges thecambium of scion and rootstock.

Cocoa’s top grafting has some advan-tages such as obtaining new plant which hasthe same trait with its parents, fast growing,early precosity, cheap, simple, well-developed

roots, and combining superior traits from itsparents. On the other hand, disadvantage ofthis method is rootstock-scion incompatibility.Difference in rootstock and scion affectscocoa production and plant growth vigour(Prawoto, 2008; Pang-Thau-Yin, 2004). Ithas been reported that survival rate of graftedscions could reach 80% in condition whereair temperature 27.7OC with relative humidity(RH) 76.2% (Prawoto, 2007). Previous studycarried out by Anitasari & Susilo (2012) hasreported grafting performance of some scionclones and root-stock family on cocoa.

ISSN: 0215-0212 / e-ISSN: 2406-9574

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Zakariyyah & Yuliasmara

PELITA PERKEBUNAN, Volume 31, Number 3, December 2015 Edition

Supply of rootstock highly depends onthe availibility of cocoa seeds, meanwhilethe availability of scions depend on numberof scion budwood which derived fromplagiotrophic branches. On the other hand,plagiotrophic branches are the places ofmostly growing cocoa pods; therefore, thebranch availability is limited by some factors.Taking of budwoods from selected superiortrees should be done either in the early or theend of rainy season. Meanwhile, taking outof budwood from productive farms shouldbe done in limited number considering that itmay affect plant production. Application ofgrafting in cocoa nursery may last for sixmonths considering that the seedlings willbe planted in the beginning of the rainy season.Seeds for rootstock are planted in June whiletaking budwood for scion material is carriedout in August. Meanwhile, recommendedminimum number of scion budwood is threebudwoods for each grafting. However, needfor budwoods especially in large quantities inlimited period is a challenge for cocoa nurseryplanters when using top grafting method.In the case of cashew plants, the numberof grafted scion budwoods affected shootlength, number of leaves and percentage

of flowers bearing fruits, as reported bySuharto (2012). Reducing budwood numberis an effort to overcome problems in limitedscion materials. The objective of this researchwas to study the effect of scion budwoodnumber in some cocoa clones on their seedlingperformances.

MATERIALS AND METHODS

The research was conducted inKaliwining Research Station, IndonesianCoffee and Cocoa Research Institute,Jember, Indonesia at an elevation of 48 mabove sea level. The soil in the experimentalsite is classified as Inceptisol, with D climatetype based on Schmidt & Fergusson clas-sification.

The layout for this study used factorialwith two factors in randomized complete blockdesign, with four replications for every treat-ment. Each plot consisted of five top graftedcocoa (Theobroma cacao L.) seedlings. Thefirst factor was cocoa clone types, namelyMCC 02 and Sulawesi 1. The second factorwas number of scion budwoods, namely 1,2, and 3 budwoods per rootstock (Figure 1).

Figure 1. Number of scion budwoods, namely three (A), two (B), one (C)

A B C



Four months old rootstock derived fromSulawesi 1 clone with open pollinated seeds.Nursery maintenance was conducted accordingto standard operational procedure, includingfertilizing, pruning and controlling of pestsand diseases.

The observed parameters were livebudwood percentage, shoot length, stem girth,stomatal conductance, photosynthesis rate,and stomatal diffusion resistance. Stomataldiffusion resistance and stomatal conductancewere observed using a leaf porometer(Decagon®, USA) referred to Zakariyya &Prawoto (2015).

Photosynthesis activity was measuredusing Photosynthesis Yield Analyzer (Mini-PAM®) which was tagged on cocoa leaves,namely third youngest, fully expanded andfully hardened leaves, because those leavesare the most physiologically active (Daymondet al., 2011). Measurement using Mini-PAM®

was conducted by tagging a detector to theadaxial leaves. Content of total chlorophyll,chlorophyll a, and chlorophyll b were measured

using SPAD-502. The equation for calculatingthe content of chlorophyll a, chlorophyll band total chlorophyll referred to the researchconducted by Netto et al. (2007). The equa-tions to calculate chlorophyll a,b and totalwere: chlorophyll a: Y = 15,5866 + 1,0338X+ 0,0679X2; chlorophyll b: Y = 30,1471 –0,4592X + 0,0270X2; total chlorophyll: Y =44,5885 + 0,7188X + 0,0933X2. Y is contentof chlorophyll (a, b, or total) and X wasSPAD-502 reading. Chlorophyll a, b and totalwere expressed in mol.m-2 unit. Data analysisused Excel and SPSS 14.

RESULTS AND DISCUSSION

Based on this research results, varianceanalysis showed that clones affected stomataldiffusion resistance and photosyntesis activity(Fv/Fm). Meanwhile, budwood numberinfluenced shoot length. However, theirinteraction affected stomatal diffusion resistanceand stomatal conductance (Table 1).

Table 1. Analysis of variance of stem girth, shoot length, total chlorophyll (a+b), chlorophyll a, chlorophyll b, stomataldiffusion resistance, photosynthesis activity (Fv/Fm) as affected by clone and number of scion budwood and theirinteraction

Budwood parameters

Stem Girth 1,86 ns 0,34 ns 0,96 ns

Shoot Length 0,01 ns 6,9 * 1,21 ns

Total chlorophyll 4,15 ns 1,22 ns 0,62 ns

Chlorophyll a 4,08 ns 1,21 ns 0,63 ns

Chlorophyll b 4,47 ns 1,25 ns 0,61 ns

Stomatal diffusion resistance 5,88 * 2,72 ns 6,55 *

Fv/Fm 21,73 * 0,14 ns 0,11 ns

Stomatal Conductance 4,34 ns 1,56 ns 5,39 *

Clones Scion budwood numbers Interaction

Note : ns (not statistically significant); * (significantly different)

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The result showed that the treatmentshad difference in life percentages of graftedcocoa seedling (Figures 2), where forMCC 02 clone was higher than Sulawesi 1clone. Rootstocks grafted with three scionbudwoods had higher life percentages thanother treatments. However, higher life per-centage was obtained in MCC 02 clonegrafted with three budwoods than Sulawesi 1clone with the same number of scionbudwood. Meanwhile, on grafting seedlings,the smallest viability was shown by Sulawesi 1clone with two budwoods of scion, namely

45%, then followed by those clones witha budwood, i.e. 55%, despite they showedno significantly difference each other. OnMCC 02 clone, grafting by using one andtwo budwoods consistently gave high lifepercentages which were 75% and 80%, respec-tively. Based on Anita-Sari & Susilo (2012)work, Sulawesi 1 was grouped in mediumviability.

Budwoods are needed as food reser-vation for early growth of seedlings. Basedon the research, number of scion budwoodaffected the success of grafting seedling

Figure 2. Performance of cocoa seedlings grafted with one budwood (A); two budwoods (B); and threebudwoods (C) of scion.

Sulawesi 1 MCC 02

1 2 3 1 2 3

Via

bilit

y, %

100

80

60

40

20

0

55.0045.00

80.00 75.00 80.00

95.00

Figure 3. Viability of Sulawesi 1 and MCC 02 clones with different the number of scion budwood.



viability. Risk of minimum budwood numberis no substituted shoots which can be main-tained when the other shoot was attackedby Phytopthora palmivora disease andHelopeltis sp., dry bud risk, and the occur-rence of other physical disorders. Helopeltisbecomes a serious problem in the processof grafting in seedling stage. Helopeltis attacksand inhibits shoot growth as a result theleaves become dry (Atmadja, 2003; Santosoet al., 2013). Bahri & Santoso (2013) reportedthat for cassava cutting needed at least twobuds. Seedlings with less than two buds wasvulnerable to drought and less viability.Therefore, special handling is neccesaryrelated to its treatment e.g. pest control,fertilization, and others to optimize theseedling vigour.

Shoot length

A single factor of clone did not signifi-cantly affect shoot length; meanwhile, numberof scion budwood significantly affected thatparameter (Table 2). The highest shoot lengthwas obtained from grafting with onebudwood. Grafting with two and threebudwoods showed significant differencecompared with one budwood. Number ofbudwoods reflects different scion length,

meaning that the more budwood numberwas, the longer scion size would be.

Waluya (2011) reported that on cassavacutting, the higher number of scionbudwoods is, the higher number of shootswill appear. Thus, the consumption of foodreservation in plant will be higher and maycompete each others. In another case, thedifferences of budwood size in grafting havebeen attributed to the change of shootconcentrations of phytohormones, such asauxin, gibberellin, absisic acid, and cytokinin(Grochowska et al., 1984; Kamboj et al.,1998; Kamboj et al., 1999; Steffens &Hedden, 1992).

Shoot Girth

According to Table 3, shoot girth wasnot different among the treatments, eitherclones or number of scion budwoods. Ingeneral, shoot girth of MCC 02 was greaterthan Sulawesi 01, even though it was notsignificantly different. Scion which is highlycompatible with rootstocks and has well graftunion will support translocation of assimila-tion, water, nutrients, hormones and enzimesto the whole seedlings. Initiation of morpho-genic scions by grafting induces morphologicalchanges on leaves, flowers and fruits due to

Table2. Effects of clones and number of scion budwoods on shoot length of grafted seedling

Sulawesi 1 11.8 a 1 14.8 a

MCC 02 11.7 a 2 8.4 b

3 12.1 ab

Clones Shoot length, cm Scion budwoods number Shoot length, cm

Note: Numbers within the same column followed by the same letter are not significantly different at 5% level according toDuncan test.

Table 3. Effects of clones and number of scion budwoods on shoot girth of grafting seedling

Clones Shoot girth (cm) Scion budwoods number Shoot girth (cm)

Sulawesi 1 1.1 a 1 1.2 a

MCC 02 1.3 a 2 1.1 a

3 1.3 a


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genetic changes which have potential ingenetic improvement (Eltayb, 2014).

In general, the number of budwoods(scion) which were grafted to the rootstocksgrew similarly. However, different numberof scion budwood did not consistenly affectshoot girth. In the field, scion growth canbe vary with the predicted size from therootstocks to which they are grafted. It maybe affected by environmental factors.Tworkoski & Fazio (2011) reported thatvarious size of apple fuji scions which weregrafted to the same rootstocks size growuniformly, but there was a modification ofxylem sap size.

According to Sukarmin et al. (2009),food reservation which is accumulated intorootstocks is formed by photosynthesisprocess and required to support the callusinitiation on graft union as well as to stimulatebudwood dormancy to break and then grow.Suharsi (2013) found that the readiness ofroot stock to be grafted on scion associatedwith the age and size of the rootstock. Theolder and larger rootstock is, the better stemgrowth stimulation will be.

Chlorophyll a, b, and total

Chlorophyll is a pigment which has amain function to support photosynthesis.Based on this research, the total chlorophyll,

chlorophyll a and b were not different sig-nificantly. Based on clone and number ofbudwood, MCC 02 has higher chlorophyllthan Sulawesi 01. The treatment grafting bythree budwoods showed the highest contentin total chlorophyll, chlorophyll a and b.Chlorophyll a is dark green color pigmentand chlorophyll b is green color pigment andthey absorb red color (Ai & Banyo, 2011).However, the function of chlorophyll inphotosynthesis process transforms lightenergy into chemical energy of ATP (adenosinetriphosphate) and NADPH (nicotinamideadenine dinucleotide phosphate + H), whichoccurs in the light reactions. Furthermore, thechemical energy used in the dark reactionsreduces CO2 into carbohydrates and O2(Salisbury & Ross, 1995).

Generally, grafting method has beenreported to be able to affect some physiologicalcharacteristics especially chlorophyll andphotoshyntesis performance (Ballesta et al,2010). Some researchers have confirmed thatcompatibility of rootstocks-scion graftingtends to enhance plant fruit quality in someways (Lee et al., 2003; Angela et al., 2008;Li et al., 2009). Chlorophyll content in muskmellon plant increased in grafting treatmentswhich had impacts on photosyntesis processand carbohydrates content (Liu et al., 2011).However, the increase in photosynthesis andcarbohydrate metabolism due to grafting hasthe potential to improve plant quality.

Table 4. Effects of clones and number of scion budwoods on total chlorophyll, chlorophyll a and b content

Note: ns = not statistically significant at 5% level according to Duncan test.

MCC 02 1 180.7 ns 132.2 ns 46.7 ns

2 170.9 ns 123.8 ns 45.4 ns

3 229.9 ns 171.0 ns 57.0 ns

Sulawesi 1 1 139.5 ns 99.1 ns 38.8 ns

2 157.2 ns 113.4 ns 42.0 ns

3 161.0 ns 116.4 ns 42.8 ns

ClonesScion budwoods

numberTotal chlorophyll

(mol.m-2)Chlorophyll a

(mol.m-2)Chlorophyll b

(mol.m-2)



Photosynthesis (Fv/Fm)

The value of photosynthesis can be ex-pressed in Fv/Fm units. The value of Fv/Fm is flouresence value which indicates themaximum photochemical efficiency ofPhotosystem II or the maximum photo-chemical energy conversion (Lamontagneet al., 2000; Heinz-Walz-GmbH, 1999). Themaximum quantum yield (Fv/Fm) can beestimated by measuring the increase influorescence yield from dark-adaptedminimal fluorescence (Fo) to maximal fluo-rescence (Fm), which is associated with theclosing of photosynthetic reaction centersduring light saturation or a photosyntheticinhibitor such as 3'-(3,4-dichlorophenyl)-1',1'-dimethyl urea (DCMU) (Cullen et al., 1979).

As presented in Table 5, photosyntheticrate showed significant difference betweenthe clones tested, however number of scionbudwood treatment has no significant effect.MCC 02 was significantly different fromSulawesi 1. The value of Fv/Fm diverselyranged from 0,2 to 0,7 (Heinz Walz GmbH,1999). However, the variations of Fv/Fmratio appeared to be related with biomassand independent assimilation. It means thatfor Fm and Fv/Fm parameters, the responseis complex, not only related to chlorophylla concentration, but also to light scatteringand re-absorption phenomenon (Ting &Owens, 1992; Büchel & Wilhelm, 1993).

The clones of cocoa grafting seedlingperformed differently for photosyntesis.

These results were in agreement with thestudy by Zheng et al. (2009) who havefound that grafting reduced damage onphotosynthetic apparatus. A similar effectwas observed when grafting tomato plantwith a salt tolerant rootstock, since itmaintained higher photochemical activityof photosystem II (He et al ., 2009).According to their research result ,Albacete et al. (2009) reported an inducedrootstock increased crop productivity towardchanges in leaf area and photosyntheticcapacity. On the other hand, photosystemII contained chlorophyll a which highlyabsorbs solar energy with 680 nm of wavelength.

Stomatal Conductance and StomatalDiffusion Resistance

Figure 4 presents a significant interac-tions between clone and number of scionbudwood to stomatal conductance parameter.Stomatal conductance is conductive ability ofstomata for gas movement from high to lowconcentration, meanwhile stomatal resistanceis the inhibition of gases to enter through thestomata (Salisbury & Ross, 1995). Drake et al.(2013) reported that stomatal characters affectedthe mechanism of gas exchange (includingCO2) in the plant especially on leaves. CO2is required in the photosynthesis process,therefore stomatal conductance will be impor-tant in obtaining an optimum performance forgrafting seedling.

Table 5. Effects of clones and number of scion budwoods on the content of total chlorophyll, chlorophyll a and b


Sulawesi 1 0.40 b 1 0.41 a

MCC 02 0.42 a 2 0.41 a

3 0.41 a

Clones Fv/FmScion budwood numberFv/Fm

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Stom

atal

con

duct

ance

, mm

ol/m

2 s

20

16

12

8

4

0

Sulawesi 1 MCC 02

1 2 3 1 2 3

b

abab

aab

a

On MCC 02 clone, rootstocks whichwere grafted to various numbers of scionbudwoods performed no significant differenceto each other. Meanwhile, several budwoodsof Sulawesi 1 grafted seedling were notsignificantly different. The highest value wasobtained on MCC 02 (17,0 mmol/m2s) graftedwith single bud scion and was differentsignificantly compare to Sulawesi 1 (12,4mmol/m2s) with single bud scion. Highernumber of scion budwood reduced stomatalconductance even though it had smallcorellation (R2=1,992). It was the contribu-tion of size of budwoods together with num-ber of scion budwood which increased theeffort to transport nutrients. Higher bud sizecaused lower transpiration and stomatalconductance.

Stomatal diffusion resistance causedreverse effect compared with stomatalconductances and this parameter was alsoaffected by interaction among treatments.The value of stomatal diffusion resistanceon MCC 02 was relatively lower thanSulawesi 1. The highest value was obtainedon Sulawesi 01 grafted with one budwood

(81,38 m2/mmol) and significantly differentto MCC 01 at the same number of graftedscion budwood. The higher the value ofstomatal resistance is, the lower the gas(included CO2) diffusion barrier will enterthe leaves, so that photosynthesis will besmaller (Zakariyya & Prawoto, 2015).

In some studies, grafting method wasreported to be able to modify xylem sap (Ballesta,2010; Johkan, 2009). Meanwhile, the com-patibility of scion-rootstock may determinewater and nutrient translocation and affectother physiological traits (Ballesta, 2010;Omid et al., 2007). Johkan (2009) foundthat grafting old sweet pepper plants resultedin poor development in their xylem connec-tions at the graft site, which results in lowstomatal resistance and water potentialcompared to younger plants. Some studiesreported that scion and number of scionbudwood affected stomatal performance,which produced higher CO2 assimilation rateand less stomatal resistance than in non-graftedand/or self-grafted plants (Yetisir et al.,2007; Rouphael et al., 2008; He et al., 2009;Zheng et al., 2009).

Figure 4. Effects of cocoa clones and number of scion budwood to stomatal conductance



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ACKNOWLEDGEMENT

The authors thank to Dr. Misnawi andDr. Agung Wahyu Susilo as supervisors. Wealso thank to Mr. Soedradjad and Mr. Herwantowho supported this research.

Figure 5. Effect of cocoa clones and number of scion budwood to stomatal diffusion resistance

Diff

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