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COMPATIBILITY STUDIES OF INTERSPECIFIC
IN VITRO MICROGRAFTING OF AGARWOOD

(Aquilaria malaccensis LAMK.)

NURITA TORUAN-MATHIUS*, JONNER SITUMORANG, DEWI RACHMAWATI &
ANIDA

Biotechnology and Plant Breeding, SEAMEO BIOTROP, Bogor, Indonesia

ABSTRACT

Aquilaria spp produced agarwood as nonwood forest production, and has high economic
value. A. malaccensis is susceptible to white rot diseases and termites. On the other hand most of the
community plantations are a mixed culture with rubber trees, oil palms and with high risk of con-
tamination causing white root diseases. Besides that, vegetative propagation by cuttings, stumping
or air layering are still very diffi cult with low percentage of growth. Th e objectives of this research
were to analyze the best suitable micrograft type, changes of SDS-PAGE protein band patterns of
compatible and incompatible micrografts with several combinations of gaharu planlets in in vitro
condition, and histology of union area between rootstocks and scion. Th e results showed that wedge
or V type was the best of the micrografs. MS medium with the addition of 3 mg/L IBA was the
best medium for gaharu planlet growth after micrografting. Acclimatization was conducted in husk
chacoal and top soil (1:1) medium and grown under plastic house of 70% shading with paranet.
Compatible combination (Ac/Am) of micrografting showed that anatomy structure of union area
is the same as anatomy structure of non micrograftd planlet. While incompatible (Gv/Am) mi-
crografting produced necrotic layer growth from pith and parenchymateous tissues of the wood in
union area along the middle of radial shoot. Recovery period of union area between stocks and
scion is initiated by callus formation from the pith and parenchymatous tissues of the wood. Callus
will diff erentiate into mature cells or tissue and become combined phloem and xylem vessels be-
tween rootstocks and scion. SDS-PAGE protein band pattern on compatible combination was the
same as plants originated from seedlings. While, incompatible combination produced new protein
bands with molecular weight around 21 and 30 kD.

Key words : Agarwood, Aquilaria spp, micrografting in vitro, incompatible micrografting, SDS-
PAGE protein, incompatible histology.

INTRODUCTION

A. malaccensis, is a famous species for production of good-quality agarwood.
Agarwood is an inducible secondary metabolite caused by interaction between defence
materials like phytoalexin of gaharu trees against compatible pathogen of cells in woody

BIOTROPIA VOL. 15 NO. 2, 2008 : 95 - 109

*Corresponding author: nurita-toruan@smart-tbk.com

tissues (Van der Plank 1978; Kunoh 1990; Rahman & Basak 1980; Parman et al. 1996).
Th is secondary metabolite resin is not secreted from trees like other resins or gum, but
accumulated in infected stems or branches. Th is phenomenon causes the white and
smooth part of wood turns to be dark and hard. Th is wood becomes heavier and fragrant
if it is burned (Hou 1960; Anonim 2002; Subansenee et al. 1985).

Gaharu is of high economic importance in Asia due to its use for the production of
incense, perfumes and traditional medicines. A. malaccensis is recognized on the IUCN red
list as critically endangered and considered to be at risk from overexploitation. All species
of Aquilaria were included on the Appendix II list of the Convention on International
Trade in Endangered Species of Wild Fauna and Flora (CITES) in 1994 (CITES 2003)
to improve control of commercial gaharu trade in all participating countries.

To prevent A. malaccensis to become an endangered species, Indonesia has compiled
a National program for sustainable agarwood production by planting of this species in
several provinces through government project and community participation (Suparman
2006). Th ese programs faced problems because A. malaccensis is susceptible to white
root diseases and termites. On the other hand, most of the community plantations are
a mixed culture with rubber trees, oil palms and with high risk of soil contamination
causing white rot diseases; A. malaccensis is a relatively slow-growing plant compared
with other Aquilaria species; Recalcitrant seed with low viability, hand–sown seeds have
germination rate (67% for those planted immediately, decreasing to 47% for those sown
after 1 week).

To solve these problems in vitro micrografting between A. malaccensis as a scion
and other species of Aquilaria or Gyrinops as a rootstock, or interspecifi c micrografting
could be used for propagation. Toruan-Mathius et al. (2006 b) reported that SEAMEO
BIOTROP has succeeded to develop tissue culture technique for propagation of A.
malaccensis, A. crassna, A. becariana and Gyrinosp verstigii from selected mother plants,
acclimatized and planted under fi eld conditions. Th ese techniques are used as a basis for
in vitro micrografting.

In vitro micrografting : Shoot-tip grafting in vitro (STG) consists of grafting, under
aseptic conditions, a small shoot tip onto a young seedling or planlet root stocks growing
in vitro. Th e technique has the following steps: rootstock preparation, scion preparation,
grafting procedure, growing grafted plants in vitro, and transferring to soil.

Possibility of micrograft less diff erentiates shoot tip tissue may also help in reducing
compatibility problems between scion and stock (Jonart 1986).

Several reports have described the application of in vitro and in vivo micrografting
that may provide several advantages such as rejuvenation of mature tissues, year round
plant production, enhanced compatibility studies and correlative relations between
root stocks and scions, make specifi c genotypic combinations to increase productivity,
and extend ecological limits of a particular plant species or cultivar to tolerate edaphic
conditions (Richardson et al. 1996; Hartmann et al. 1997; Estrada-Luna et al. 2002;
Sobhana et al. 2001; Sanjaya et al. 2006). Toruan-Mathius et al. (2006a) have succeeded
to use in vitro and in vivo interspecifi c micrografting of Cinchona ledgeriana as a scion
onto C. succirbura as a rootstock to avoid white root disease. Under fi eld condition, in
vitro micrografting grows faster than that of in vivo grafting.

Da Costa et al. (1991) observed that production of plants grafted on Robusta
coff ee was three to four times higher than that of nongrafted plants. In addition it is

BIOTROPIA VOL. 15 NO. 2, 2008

also used to increase resistance to soilborne parasites or soil-borne diseases caused by
pathogens such as Fusarium oxysporum. Grafting is also commonly used for fruit trees and
in silviculture (i) to improve tree adaptation to unfavorable soil or climatic conditions, (ii)
to enhance water and nutrient uptake, (iii) to increase plant vigour and extend duration of
economic harvest time, and (iv) to shorten the breeding period by limiting the breeding
objective for resistance to soil-borne diseases and nematodes in root stock. Th ese fi ndings
suggest that the existence of this technique is an eff ective means of rapid and true-to-type
multiplication of desired A. malaccensis genotypes.

Th e objectives of this study were: (i) to determine the appropriate and reliable
micrografting techniques for agarwood planlets; (ii) to determine survival, growth,
rootstock suckering, and short-term compatibility of A. malaccensis scion on several species
of rootstocks under in vitro conditions, and (iii) to characterize graft incompatibility
based on histological and biochemical aspects of grafting.

MATERIALS AND METHODS

Th e study consisted of rootstock preparation, scion preparation, (a) In vitro
micrografting, (b) analysis of compatibility by histology and (c) electrophoresis SDS-
PAGE protein of stem from scion.

Plant materials
A. malaccensis B23 line used as scions were micropropagated following techniques

established by Situmorang (2000). Axillary buds excised from young planlets (3-5 cm
long) were initially used as explants and induced to develop shoots using Murashige and
Skoog (1962) basal salt formulation medium adjusted at pH 5.7 and supplemented
with 5.0% sucrose and 0.6% bacto agar. BA (6-benzyl aminopurine) of 1.0 mg/L was
added for shoot induction and proliferation cultures. Adventitious roots developed in MS
medium with the addition of 3 mg/L IBA (Indole Butyric Acids). Planlets of vigorous
A. crassna, A. fi laria and Girynops verstigii as stocks were propagated by tissue culture the
same as for planlet scions.

All cultures were grown in a culture room with light of 100 μmol m-2 s -1
photosynthetic photon fl ux density (PPFD) cool white fl uorescent lamps at planlet level
and photoperiod of 16 h. Temperature was maintained at 27 ± 2o C.

Micrografting
Under aseptic conditions, shoots of uniform length and diameter were selected

from in vitro culture and used as rootstocks and scions in micrografting. Stainless silver
blades mounted on a handle were used for cutting the plant material. Freshly picked
shoot tips and in vitro regenerated shoot tips were used as microscions. To prepare the
initial scions of the micrografts, the leaves from those portions were eliminated, and four
types of grafting were made in each of their basal parts.

For micrografting, the following method was used. When the stem axis of the
plantlets reached a length of about 2 cm the upper 1.0 cm of the tips were excised and
used as scion, while remaining portion was used as rootstock. In the fi rst experiment four
types of grafting viz., a) wedge (V), (b) horizontal - cut grafting, (c) slant-cut grafting (/)

Interspecifi c in vitro micrografting of agarwood – N. Toruan-Mathius et al.

and (d) cleft grafting (I) (Fig.1) were made in the rootstock and scion. Th e scions were
placed in the incisions made in the rootstock with both the cut surfaces in good physical
contact (Fig. 1). Th e stock and scion were held together at the point of graft with sterile
(1x1 cm) light aluminum foil.

For optimizing the method of micrografting in vitro plant materials and as scion
only B 23 and A. crassna as a rootstock were used. On the other hand, for compatibility
studies three clones of A. malaccensis as scion and three species as rootstocks were used,
with the best method of micrografting, and as a control the same species of scion and
rootstocks were used. Th e percentage of successful and unsuccessful grafts was determined
30 days after grafting and in vitro culture.

All planlets of graft treatments were cultured in MS medium + 3 mg/L IBA for
rooting. After three months the growth of scion as a result of graft union was observed.
For incompatibilities studies, the combination of scion-stock i.e. A. malaccensis as scion
and A. fi larial, A. crassna, Gyrinops verstigii as stocks could be used, while as control the
same species of scions and rootstocks were used. Cultures were incubated in light culture
room (16 hrs/days) with Rh 70-80%.

a b c d

(i)

(ii)

(iii)

(iv)

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Figure 1. In vitro micrografting between scion of A. malaccensis planlets with rootstocks of several species
of Aquilaria sp and G. verstigii, using (i) wedge, (ii) horizontal, (iii) slant, and (iv) cleft grafts.
a. micropropagated shoots, b. rootstock, c. scion, and d. methods of grafts.

Growth analysis
Th e in vitro micrografting success rate was determined 6 weeks after grafting

by recording the number of scions still alive out of all individual treatments. Number
of leaves were also recorded and measured for each sample, 2 months after the date of
micrografting, and recorded continuously for every month during 8 months observations.
At the end of the experiment, all plants were carefully removed to study their root systems.
For fi ve plants from each graft combination and the control, the stem was cut crosswise
for macroscopic observation of graft union or analysis of compatibility.

Experimental design
Combination of stock-scion viz.: A.malaccensis/A.malaccensis (control 1), A.

crassna/ A. crassna (control 2) , A. fi laria/ A. fi laria (control 3); A. malaccensis/ A. crassna;
A. malaccensis/ A. fi laria was used. Experiments used Completely Randomized Design of
1x3 units with 15 replicates.

Analysis of compatibility
Analysis of compatibility between scion and rootstocks studies by histology

and electrophoresis SDS PAGE protein of graft union, were conducted for in vitro
micrografting.

Histology of graft
A histological technique was used to observe the graft union of every combination

of root stock/scion. Th e graft union was cut into segment of 2 cm long and microtome
was used to cut 25 μm slices from the segments. Th e samples were fi xed for 24 h in
solution of FAA and then dehydrated in an ethanol series (50-70-80-90-95-100-100%)
for 30 min. each. Safranin (2%) was added in 70% ethanol and fast green (1%) in 100%
ethanol for 15 seconds. Th e slices were placed in a solution of 1 absolute ethanol: 1 xylene
for 30 seconds and then in pure xylene (2x for 30 min. each), before being examined
under the microscope (Esau 1965).

Electrophoresis SDS-PAGE protein
Total protein was extracted from scion shoot (2 cm above graft line) by frozing

in liquid nitrogen and stored at -40o C. Plant tissues were homogenized in a mortar in
the presence of liquid nitrogen and extracted using buff er containing 5 mM Tris HCl,
pH 8, 500 mM NaCl, 2 mM ascorbic acid, 0.5 mM phenylmethylsulphonyl fl uoride
and 1 mM dithiotreitol (Laemmli 1970). Th e homogenate was heated at 80o C for 10
min. and centrifuged at 17.000 g. Supernatant were thawed and heat stable proteins were
quantifi ed by the binding-dye assay (Bradford 1976). Protein was separated by 10-15%
polyacrilamide gradient gels. Heat stable proteins 5-15μg were loaded in each well with
discontinuous buff er systems (Laemmli 1970) using a Mini protean 3-electrophoresis cell
(Bio-Rad, Hercules, CA, USA). Gel lanes were loaded with equal amounts of perceptible
counts; low molecular weight protein standards (Bio-Rad) were run as size markers.

Electrophoresis run with constant current of 75 mA was applied per gel. During
electrophoresis the voltage increased from about 300 to 600 V. A 0.025 M tris-glycine
buff er (pH 8.6) was used as the electrode buff er. When the tracking dye had migrated to
the bottom of gel (after approximately 3.5 hrs) electrophoresis was stopped. Gels were

Interspecifi c in vitro micrografting of agarwood – N. Toruan-Mathius et al.

100

stained with Commasie brilliant blue. After staining gels were fi xed in solution containing
methanol, glacial acetic acid and distilled water with the ratio of 4:1;15 (v/v/v) for 15
min. and photographed, then gels were wrapped with cellophane for documentation.

Th e eff ect of stock on scion was studied by the formation of new protein band or
disappeared protein band compared with control (non micrografting and micrografting
scion-stock with the same species).

Statistical analysis
All data were subjected to one-way analysis of variance (ANOVA) using SAS

version 6.12 (SAS Institute 1996). For categorical data, the normality of residuals and
homoscedasticity were verifi ed to fulfi ll the requirements for ANOVA. Duncan’s multiple
range test was used for mean separation. Th e diff erences were considered signifi cant at P
≤ 0.05.

RESULTS AND DISCUSSION

In vitro micrografting
Th e type of graft and nature of support signifi cantly infl uenced the success of

micrograft. Wedge or V graft gave the most successful micrografts followed by cleft graft
method (Tabel 1). V graft may provide a better surface for the contact of stock and scion
providing good cambial joining at the graft union. It was shown that V type of micrograft
also gave highly signifi cant diff erence on grafting success, except on the fi rst week of
incubation because of callus formation (Table 2). A good result was obtained with
micrograft V type, which gave faster growth compared to cleft graft, caused by V graft
which have high levels of graft union (Table 2). Micrografting of gaharu planlet with V
graft in combination between rootstocks and scion Ac/Am and Am/Am showed the
same vigour (Fig. 2).

(a) (b)

Figure 2. Micrografting of gaharu planlet with V type in combination between rootstocks and scion:
(a) Ac/Am, and (b) Am/Am. Ac – Aquilaria crassna; Am – Aquilaria malaccensis

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Table 1. Effect of micrografting techniques on union and growth of scion

Method of grafting Number of grafts Grafting success (%)

Wedge (V graft) 25 98,5 a
Horizontal 20 25.6 c
Slant 25 28,6 c
Cleft grafts 25 80,7 b

Estrada-Luna et al. (2002) reported that the best method for in vitro micrografting
of prickle cactus (Opuntia spp) were horizontal and wedge grafts. Th e fi rm contact
between rootstock and scion is extremely important at the graft junction to get fusing
between them and callus formation. Lukman et al. (2005) found that micrografting of
two diff erent species (mundo and manggis) with V type, gave 76% succesful graft rate
when the shoot tips were used directly as microscions in in vitro studies. Oda (1995)
said that micrografting with V type will gave a fi rm and faster union between stocks and
scion.

Tirtawinata (2003) explained that the success rates of micrograft are highly
dependent upon the cambium union of rootstocks and scion. Partial contact of rootstock
and scion cambium will contribute to unsuccesful joining. Th ese cases are described as
“translocated” graft incompatibilities (Estrada–Luna et al. 2002) According to Hartmann
et al. (1997) callus formation occurred as parenchym cells in a process of wound
recovery. Th ese callus will diff erentiate into cambium, xylem and phloem. Th ese vessels
are used for transportation of nutrition and photosyntate. Kala et al. (2002) reported
that in vitro micrografts rubber begin to grow weeks after micrografting. Rifa’i (2003)
found callus formation up to three months after micrografting.

Tabel 2. The effect of micrograft types on leaf number of 1-8 week- old planlets after micrografting.

Combination and
type of micrograft

On weeks ....
1 2 3 4 5 6 7 8

Ac/Am–V 0 0.4 a 1.56 a 2.28 a 3.00a 3.88 a 4.56a 5.08a

Ac/Ac-L 0 0.71b 1.20b 1.58b 1.72b 2.95b 2.14b 2.23b

Am/Am-V 0 1.27a 2.24a 3.00b 3.84 a 4.08a 4.92 a 5.48a

Am/Gv-L 0 0.85b 1.23b 2.08b 2.84b 3.52 b 4.20b 4.84b

Ac/Ac-V 0 1.14a 2.00 a 2.60a 3.,52a 4.,00a 4.68,a 5.32a
CL/CL-L 0 0.71b 1.16b 2.00b 2.40b 3.24b 4.00b 4.48b
Respons tn * * * * * * *

Explanation ns : not significantly ; *.Significantly different. Numbers followed by the same letter in

the same column are not significantly different according to Duncan test P < 0,05

Interspecifi c in vitro micrografting of agarwood – N. Toruan-Mathius et al.

102

Acclimatization
Th e average of planlets survived after acclimatization in husk charcoal : sterile

top soil (1 : 1) after one month incubation was 90% (Tabel 3). Gunawan (1992)
reported that planlet from in vitro culture i.e. has wax layer on undeveloped cuticula,
limited shoot lignifi cation, number of leaves palisade cells very low, undeveloped xylem,
disfunction of stomata caused high transpiration it caused the scion become sensitive to
evapotranspiration, attack of fungi and soil bacteria, and also to high light intensity. Th at
is why planlets should be acclimatized before transfering into the fi eld.

Table 3. Survival percentage of gaharu planlet after acclimatization two months grown in plastic
house.

Combination and Graft type % of survived planlet
Ac/Am -V 92 b
Ac/Ac -V 90 b
Am/Am -V 90 b
Ac/Am -cleft 60 a
Ac/Ac -cleft 66 a
Am/Am -cleft 65 a

Explanation : Numbers followed by the same letter in the same column are not significantly different
according to Duncan test P < 0.05

Histology of micrografts
Th e results showed that anatomy structure of gaharu shoot is the same as

anatomy structure of plantlet shoot of another Dicotyledone plant which consisted of
phloem, cambium and xylem. Cambium was radially in between of phloem and xylem
vessels. Th e inner part of cambium develops into xylem vessels which function as a
transportation of water and nutrition from roots to upper parts. Outside the cambium is
phloem which functions as a transportation of photosyntate to ground parts of plant.

Our observation revealed fi ve development stages during graft union formation:
development of necrotic layer, proliferation of callus bridge at the graft interface,
diff erentiation of new vascular cambium, restoration of new vascular tissue, and restoration
of the continuity of the epidermis at the graft union. Unions between parenchymatous
tissues have been confi rmed 2-3 weeks after grafting. Fifteen days after grafting parenchyma
unions occur in all micrografts that have prospects of further development. Only cells
newly formed after grafting are able to unite.

Th e fi rst unions occur between cells originating from tissues outside the cambial
region. In side-slit grafts with radial incision in the bark of the rootstocks, the fi rst union
developed between callus from the rootstock cambial region in the barks fl ap, and callus
from cortex, phloem rays and pith of the scion. Trial incision on the rootstock unions also
occurs at the incision face in a way similar to that of veneer side grafts. Union of newly
formed cells may develop independently of the kind of originial tissues (Fig. 3a & 3b).

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Figure 3. Histology union area of compatible and incompatible micrograft
(a, b & c) radial cross section of stem at union area.
(a). Non micrografted stem, (b) union area of compatible micrograft,
(c) union area of incompatible micrograft
(d & e) longitudinal cross section of stem union area.
(d) union area of compatible micrograft, (e) union area of incompatible
micrograft. (a). 1- cambium, 2- pericycle, 3- cortex
(e) 1- necrotic cells ; 2- callus; — Bars (40 μm)

Histological investigations concluded that callus bridging between scion and the
rootstock was completed three weeks after micrografting and that both partners took part
in the development of the callus. Diff erentiating xylem elements within the callus bridge
were observed by the fourth week but no bridging of the graft union was achieved at this
stage. Noticeably, each partner established new vascular system outside the callus bridge,
directed towards the others. Hartmann et al. (1997) explained that callus is a group of
parenchym cells which has developed around wounded tissues. Growth of parenchym
cells is a initial phase of callus formation in union area of stocks and scion. Parenchym cell
has high ability to divide and develop into mature cells. Cell development and anatomy
of planlet will change, and its function of each tissue/cells one to another is also diff erent.
Undiff erentiated tissues have diff erent functions compared to diff erentiated tissues.
Callus will diff erentiate into mature cells or tissue and become a combined tissue between
rootstocks and scion. Callus growth was found from the combination of Gv/Am and
Gv/Amc (Fig. 3 b, c & d) but diff erent with the seedling or Am/Am (Fig. 3). It was
shown that the rootstock aff ected the growth of the scion, changes the morphology and

Interspecifi c in vitro micrografting of agarwood – N. Toruan-Mathius et al.

104

anatomical structure of the scion. In contrast, the scions have little eff ect on rootstock
growth.

Mismatch of phloem fi ber bundles has an important eff ect on graft success (Fig
3e). On the whole, the rootstocks produce a large volume of callus before union than the
scions but leaf traces adjacent to cut surfaces of the scions cause locally larger formation of
callus. Rays that have been cut far from the phloem usually form more callus than those
which have been cut close to the cambium. Th e boundary between phloem and cortex is
a very active zone of growth.

Th e cambial region plays a subordinate role as a callus producer in veneer side
grafts in the rootsocks of V graft. However, the loose bark from the wood in the cambial
region and the cells produce vigorous callus formation (Erea et al. 2001). Th is process is
initiated by ray cells, but soon the major portion of the cambial cells participate if they
are undamaged by grafting. Callus formation from the pith and from parenchymatous
tissues of the wood occurs only in union area . Th e pith of the scion is particularly active
when cut.

Since the cambium rootstocks had already entered an active stage at grafting and
the rootstocks have water and nutrients supply higher than that of the scion, its cambium
will be able to deposit several new rows of tracheids up at the time when union is possible.
Th is means that the cambium is forced to move from scion cambium which has been
placed in front, but which is catching another cambium that has been placed outside (Fig
3).

Th e fi rst unions of vascular tissues in veneer side grafts are often found between
the stock fl ap and tissues situated on the short cut surface of the scion leaf traces which
appear to play an important role in the establishment of vascular connections. Th e
cambial unions in the innermost corner of side slit grafts become complex depending
on the mutual position of the tissues of the grafts components. It is not necessary for the
space between the wood surfaces of the graft components to be fi lled with callus tissues,
above the uppermost point of contact with the scion. Th us the wound caused is callused
over from all sides in the same manner as in cut branch.

Rifa’i (2003) found that callus formation at union area still continues up to three
months after micrograft. Tirtawinata (2003) reported diff erentiation process of new
cambium occurred after three months of micrografting. After fourty days to 6 months of
union area will make apple micrografts become fi rm (Richardson 1996).

Estrada-Luna et al. (2002) explained that compatible micrograft growth of scion
planlet is very vigourous, while the incompatible one is dwarfi sh. In walnut (Junglans
regia) union of vascular tissues have been confi rmed after about three weeks in well-
matched grafts. When the matching has not been well done, union may require 5-6
weeks. In well-matched grafts it is common that divisions in the cambial region are able
to serve in the union of vascular tissues almost at once. Only a small number of short,
irregular cells are then formed. When the cambial region is located further apart, they
spread through intermediate parenchyma tissues toward each other by joining from one
cell to the next.

Heteroplastic or interspecifi c micrografting is used in fruit tree breeding to produce
early and abundant fl owering small trees. Th e same eff ect also occurs when grafting forest
trees for seed orchards. In order to avoid nematode damage to roots of Coff ea arabica L. in
Latin America, a common practice is to apply interspecifi c grafting on C. canephora var.

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Robusta (Pierre) rootstocks. Villain et al. (1996) observed that with heavy Pratylenchus
sp, infestations, production of Arabica plants grafted on a C. canephora var. Robusta
(2n=2x=44) rootstock was four times higher compared to nongrafted plants.

Electrophoresis SDS - PAGE protein
Th e results showed that leaf protein of scion tested has molecular weight of 14

- 45 kD. Generally SDS-PAGE protein band patterns of scion are the same with planlet
control or non micrografting, except from Am/Amc and Am/Gv. Two of the small
protein molecules, 21 and 30 kD were found only in these combinations (Fig. 4). Th ere
are several basic strategies for identifying stocks/scion interactions at the biochemical
level.

2
0
0
1
1
6

9
7

2
0
0
1
1
6

9
7

k k

Figure 4. Electrophoregram leaf protein of scion as a result of micrografting in vitro. M. Marker;
1. Am; 2. Amc; 3. Gv; 4. Am/Am; 5. Amc/Amc; 6. Gv/Gv; .Am/Amc; 8. Am/Gv; 9. Amc/
Gv

Th e most important step in identifying a biochemical basis for stocks/scion
interaction is verifying that there is caused- and- eff ect- relationship between the present
particular proteins.

New protein formation in incompatible rootstocks/scion may be regulated by
proteins because these molecules constitute the machinary of cell metabolism. When
examining a development change of varietal diff erence in a metabolic process, the cause
of the change or diff erence is most commonly a change in the presence, stability, or
activity of particular proteins.

Ji-Zhong et al. (2002) reported that incompatible micrografting of apple caused
an increase in POD and IOD enzymes activities. Th ese enzymes controlled auxin
translocation from root to upper parts of plant. Holbrook et al. (2002) found that on
compatible micrografts there was a positive relationship between rootstocks and scion. Th ese
conditions have been correlated with nutritions distributions, translocation of water and
nutrition, and regulation of hormon transportation. Raghothama (1999) explained that in

Interspecifi c in vitro micrografting of agarwood – N. Toruan-Mathius et al.

M 1 2 3 4 5 6

106

compatible micrograft of Arabidopsis the relationship of source and sink between stocks
and scion is normal. Incompatible micrografts of peach and plum planlet aff ected
protein and amino acids content of the whole tissues. Bertrand and Etienne (2001)
said that the synthesis of a conserved set of proteins, heat shock proteins, in response to
water stress caused inhibition of translocation of nutrient and water. Toruan-Mathius
et al. (1999) found that variations of protein band patterns in union area of micrograft
rubber plant are caused by diff erent interactions between micrograft combinations. Erea
et al. (2001) explained that micrograft can be used as early detection of compatible and
incompatible combination of rootstocks and scion in plants propagated by micrografts. It
was clearly distinguishable that protein banding patterns signifi cantly have a relationship
with the success or failure of grafting.

In vitro micrografts usually fail due to incompatibility between stock and scion,
oxidative browning of cut surfaces, poor contact or poor development of the root
system (Toruan-Mathius et al. 2006a; Moor 1991). Sobhana et al. (2001) studied about
physiological and biochemical aspects of stock-scion interaction in Hevea brasiliensis
and found that assimilation rate of scion is being infl uenced by the rootstock. Th e
considerable CV observed between the individual plants within clone in total soluble
sugars, reducing sugars, phenol and amino acid contents, also indicated the existence
of stock-scion interaction. Toruan-Mathius et al. (2006a) reported that incompatibility
in interspecifi c micrografting between Cinchona ledgeriana and C. succirubra showed
formation of 21 and 30 kD protein and formation of stone cells in a union area.

CONCLUSIONS

Wedge or V graft was the best micrograft method for gaharu planlets, cultured
in MS medium with the addition of 3 mg/L IBA, acclimatized in medium of husk
chacoal : top soil (1:1) and incubated in plastic house.

Histology structure of compatible micrografting showed that shoot union area
was the same as structure anatomy of seedlings, while histology structure of incompatible
micrografting has a union area and necrotic layers along radial parts of shoot.

Union of vascular tissues have been confi rmed after about three weeks in well-
matched grafts, and able to serve in the union of vascular tissues almost at once. Only a
small number of short, irregular cells are then formed.

Compatible combination (Ac/Am) of micrografting showed that histology
structure of union area is the same as histology structure of nonmicrografted planlet.
While incompatible (Gv/Am) micrografting produced necrotic layer growth from pith
and parenchymateous tissues of the wood in union area along the middle of radial
shoot.

Protein pattern in compatible combination is the same as in seedlings. While
protein pattern of incompatible combination produced a new protein band with
molecular weight around 21 and 30 kD.

BIOTROPIA VOL. 15 NO. 2, 2008

107

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